Conventional Culture-based Methods Research Articles

Emerging Microtechnologies and Automated Systems for Rapid Bacterial Identification and Antibiotic Susceptibility Testing.

Rapid bacterial identification (ID) and antibiotic susceptibility testing (AST) are in great demand due to the rise of drug-resistant bacteria. Conventional culture-based AST methods suffer from a long turnaround time. By necessity, physicians often have to treat patients empirically with antibiotics, which has led to an inappropriate use of antibiotics, an elevated mortality rate and healthcare costs, and antibiotic resistance. Recent advances in miniaturization and automation provide promising solutions for rapid bacterial ID/AST profiling, which will potentially make a significant impact in the clinical management of infectious diseases and antibiotic stewardship in the coming years. In this review, we summarize and analyze representative emerging micro- and nanotechnologies, as well as automated systems for bacterial ID/AST, including both phenotypic (e.g., microfluidic-based bacterial culture, and digital imaging of single cells) and molecular (e.g., multiplex PCR, hybridization probes, nanoparticles, synthetic biology tools, mass spectrometry, and sequencing technologies) methods. We also discuss representative point-of-care (POC) systems that integrate sample processing, fluid handling, and detection for rapid bacterial ID/AST. Finally, we highlight major remaining challenges and discuss potential future endeavors toward improving clinical outcomes with rapid bacterial ID/AST technologies.

A Minireview of the Methods for Listeria monocytogenes Detection

Listeria monocytogenes (LM) is recognized as an opportunistic, foodborne pathogen that leads to the disease listeriosis. Although the incidence of listeriosis is low, listeriosis has a high mortality rate. LM can survive the most common stresses present during food processing steps, and it causes contamination in many food products. Consequently, most countries have endorsed strong restrictions on LM in food products, especially in ready-to-eat products. Conventional culture-based methods are currently the gold standard for testing, but their inefficiency can no longer meet the needs of batch inspection. As a result, a sensitive, fast, and reliable method for LM detection is required. There are many rapid detection methods for LM that are based on different principles, and an overview of the methods of LM detection is addressed here, with an emphasis on chromatographic, immunological, and aptamer-based techniques. Additionally, the prospect of developing novel LM detection methods is discussed.

Evaluation of lytB Gene for Detection of Streptococcus pneumoniae in Isolates and Clinical Specimens by Real-Time PCR

Background: Streptococcus pneumoniae is a causative agent of morbidity and mortality worldwide. Diagnosis of pneumococcal infection includes conventional culture-based and molecular methods. Differentiation of S. pneumoniae from other mitis group streptococci is not reliable. Objectives: We aimed to evaluate the efficacy of lytB gene along with lytA gene for detection of S. pneumoniae in isolates and clinical samples using conventional and real-time PCR methods. Methods: In this cross-sectional study, a total of 560 clinical specimens were collected from patients during February-September 2015. The samples were cultured on 5% sheep blood agar and suspected colonies were identified by biochemical assays. The antibiotic susceptibility test was performed by disk diffusion and serial microdilution methods. 46 culture-negative and 46 culture-positive samples were examined to evaluate the presence of lytA and lytB genes using conventional and real-time PCR methods. Results: A total of 46 (8.2%) isolates of S. pneumoniae were identified in suspected specimens. 52% (24/46) of isolates exhibited multiple drug resistance (MDR). All 46 isolates contained both lytA and lytB genes. Real-time PCR assay detected both genes with low CT values in culture-positive samples. Among culture negative specimens, one sample was positive for both the genes. Conclusions: The lytB is similar to lytA in sensitivity for diagnosis of S. pneumoniae in isolates and clinical samples based on both the molecular methods. The results confirmed the applicability of real time PCR based on lytB genes for detection of S. pneumoniae.

A Multiplex RT-PCR Assay for S. aureus, L. monocytogenes, and Salmonella spp. Detection in Raw Milk with Pre-enrichment.

This study firstly developed a multiplex real-time PCR (RT-PCR) technique combined with a pre-enrichment step to simultaneously detect Staphylococcus aureus (S. aureus), Listeria monocytogenes (L. monocytogenes) and Salmonella spp. in raw milk and the dairy farm environment (feces, soil, feed, water) in one reaction. Brain heart infusion (BHI) broth was selected for the enrichment step to increase the density of the target bacteria by using an incubation of 4 h before multiplex RT-PCR. The results showed that the detection limit of the multiplex real-time assay was approximately 102 CFU/mL for pure cultures and artificially contaminated milk without enrichment, while 12, 14, and 10 CFU/25 mL, respectively, for S. aureus, L. monocytogenes, and Salmonella spp. after pre-enrichment. The newly developed multiplex RT-PCR assay was applied to 46 dairy farm environmental samples and raw milk samples covering a wide variety of sample types. The results demonstrated that the multiplex RT-PCR assay coupled with the BHI enrichment broth was suitable for the simultaneous screening of S. aureus, L. monocytogenes, and Salmonella spp. in the pasture environment and in raw milk. The multiplex RT-PCR assay clearly and successfully shortened the total detection time and reduced labor compared to conventional culture-based methods for testing natural samples.

Prevalence of Salmonella in poultry processing environments in wet markets in Penang and Perlis, Malaysia.

Aim:The aim of this study was to determine the prevalence of various Salmonella serotypes in chickens, carcass contact surfaces as well as environmental samples collected from wet markets and small scale processing plant.Materials and Methods:A total of 182 poultry and environmental samples were collected at random on separate occasions from wet markets and small scale processing plant, during the period of October 2014 to July 2015 in Penang and Perlis, Malaysia. The samples were analyzed for the presence of Salmonella using ISO 6579:2002 conventional culture-based method. Presumptive Salmonella colonies were subjected to various biochemical tests (such as triple sugar iron and lysine iron test), serologically confirmed using polyvalent O and H antisera and further serotyped at Public Health Laboratory, Ministry of Health, Perak, Malaysia.Results:Salmonella serotypes were isolated from 161 out of 182 samples (88.46%) with 100% prevalence in the whole chicken carcass and chicken cuts - as well as transport crate, cage, drum, knife, chopping board, display table, floor, bench wash water, wash water, and drain water. Salmonella was isolated from 91.67%, 83.33%, and 66.67% of defeathering machines, drain swabs, and apron, respectively. 17 serotypes were isolated in this study with Salmonella Albany (57/161), Salmonella Corvallis (42/161), and Salmonella Brancaster (37/161) being the predominant serovars.Conclusion:The most carcass contact and environmental samples collected along the wet market chicken processing line were consistently contaminated with Salmonella. This indicates that Salmonella has established itself in poultry processing environments by colonizing the surfaces of the equipment and survives in these environments by establishing biofilms. Our results highlight the need of implementing strict hygiene and sanitation standards to reduce the incidence of Salmonella. The prevalence of Salmonella in poultry can be reduced effectively by identifying and eliminating the sources and contamination sites during slaughter and processing of poultry.

Broad-Range Detection of Microorganisms Directly from Bronchoalveolar Lavage Specimens by PCR/Electrospray Ionization-Mass Spectrometry.

The clinical demand on rapid microbiological diagnostic is constantly increasing. PCR coupled to electrospray ionization-mass spectrometry, PCR/ESI-MS, offers detection and identification of over 750 bacteria and Candida species directly from clinical specimens within 6 hours. In this study, we investigated the clinical performance of the IRIDICA BAC LRT Assay for detection of bacterial pathogens in 121 bronchoalveolar lavage (BAL) samples that were received consecutively at our bacterial laboratory for BAL culture. Commensal or pathogenic microorganisms were detected in 118/121 (98%) BAL samples by PCR/ESI-MS, while in 104/121 (86%) samples by routine culture (P<0.01). Detection of potentially pathogenic microorganisms by PCR/ESI-MS was evaluated in comparison with conventional culture-based or molecular methods. The agreement between positive findings was overall good. Most Staphylococcus aureus-positive PCR/ESI-MS results were confirmed by culture or species-specific PCR (27/33, 82%). The identity of Streptococcus pneumoniae could however be confirmed for only 6/17 (35%) PCR/ESI-MS-positive samples. Non-cultivable and fastidious pathogens, which were not covered by standard culture procedures were readily detected by PCR/ESI-MS, including Legionella pneumophila, Bordetella pertussis, Norcadia species and Mycoplasma pneumoniae. In conclusion, PCR/ESI-MS detected a broad range of potential pathogens with equal or superior sensitivity compared to conventional methods within few hours directly from BAL samples. This novel method might thus provide a relevant tool for diagnostics in critically ill patients.

PCR-detected fungal infection is associated with fatal outcomes in cirrhotic patients with spontaneous peritonitis

Background & aimsThe rate of mortality from Spontaneous Peritonitis (SP) in cirrhotic patients is still high despite the development of new antibiotic treatments and intensive hospital care. The coexistence of spontaneous fungal peritonitis (SFP) is almost entirely ignored health problem, because it is difficult to be diagnosed at an early stage by conventional culture-based methods. Therefore, this study was designed to employ PCR-based method in evaluating the prevalence of fungal infection in cirrhotic patients with peritonitis who failed to respond to the recommended therapy and to determine its association with in-hospital mortality. Subjects and MethodsA total of 80 cirrhotic patients admitted to the hospital with ascitis (June-2013 to April-2015) were followed in this study. ResultsDuring hospitalization, 23 (42%) of patients had died although they received guideline-driven treatment. The demographics, clinical, hematological, and biochemical data were similar in both mortality and survival groups. However, the incidence of fungus infection was the only significantly elevated parameter in the mortality group than in the survival group (7/23;30% vs.0/32;0%, P=0.0012). This fungal infection was significantly associated with SP drug resistance development (P=0.007). Intriguingly, all the cases of fungal infection were detected by PCR-based method while culture-based diagnosis was able to detect the fungal infection in only 4 of these cases indicating a diagnostic sensitivity of 57%. ConclusionOur results reflect a strong association between SFP and in-hospital mortality in cirrhotic patients with SP that may offer a coherent explanation for the antibiotic treatment failure in such patients. Prompt PCR-detection and antifungal coverage is warranted in these cases.

Supramolecular Conjugated Polymer Materials for in Situ Pathogen Detection.

Cationic poly(fluorene-co-phenylene) derivative (PFP-NMe3+) forms a supramolecular complex with cucurbit[7]uril (CB[7]), which could be reversibly disassembled by amantadine (AD) to release PFP-NMe3+ due to the formation of more stable CB[7]/AD complex. The cationic PFP-NMe3+ is an amphiphilic structure and could bind to negatively charged membrane of pathogen by multivalent interactions. Upon the formation of PFP-NMe3+/CB[7] complex, the CB[7] could bury the side-chain alkyl groups and decreases the hydrophobic interactions of PFP-NMe3+ on the surface of pathogens; thus, PFP-NMe3+ exhibits different interaction modes with pathogens before and after assembly with CB[7]. The PFP-NMe3+/CB[7] supramolecular complex could be explored as optical sensor for simple, rapid, and in situ detection and discrimination of multiple pathogens by taking advantage of optical signal changes of PFP-NMe3+/CB[7] complex before and after disassembly by AD on the pathogen surfaces. The new sensor can realize in situ detection and identification of Gram-negative bacteria (E. coli, P. aeruginosa), Gram-positive bacteria (B. subtilis, S. aureus, E. faecalis), and the fungi (C. albicans, S. cerecisiae) and can also discriminate different strains of the same species. Blend samples of these pathogens could be identified successfully as well. In comparison with conventional blood culture-based pathogen assay methods that require at least for 24 h, the PFP-NMe3+/CB[7] complex only needs 2 h (including pathogen culture, pathogen harvest by centrifuging, and optical assay procedures) to stratify diverse pathogen types and also does not require specific biomarkers or cell labeling.

Rapid screening for group B Streptococcus in near-term pregnant women by Granada™ biphasic broth

Background: Prenatal screening for group B Streptococcus (GBS) colonization can reduce the incidence of neonatal GBS infections. We aimed to improve the screening-based approach of GBS in a limited resources antenatal care clinic by using Strep B Granada™ Biphasic Broth.Methods: This study included 80 pregnant women between 35 and 37 weeks of gestation, who attended the antenatal care clinic of Kasr El-Aini University Hospital from November 2013 to January 2014. Two high vaginal swabs were collected, then transported using Amies transport medium. One vaginal swab was processed by conventional culture-based methods on 5% sheep blood agar plates. The other swab was immersed in 3 mL selective enrichment broth (Granada™ Biphasic Broth bioMérieux).Results: Among 80 pregnant women, GBS was detected in 9 (11.25%) of the studied cases within 18–24 hours. Detection of orange-red colonies in GBS Granada broth was 100% specific for the presence of beta-hemolytic group B streptococci.Conclusion: Using Granada biphasic broth media was easy, affordable and shortened the turnaround time needed for the detection of GBS by conventional culture methods. Routine screening of pregnant women for vaginal GBS colonization by Granada™ Biphasic broth would allow properly timed prenatal antimicrobial prophylaxis to prevent possible neonatal infections.

Multiplex identification of drug-resistant Gram-positive pathogens using stuffer-free MLPA system.

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture-based methods require relatively longer time to identify drug-resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug-resistant Gram-positive pathogens, we developed a method by using stuffer-free multiplex ligation-dependent probe amplification (MLPA) coupled with high-resolution CE single-strand conformation polymorphisms (CE-SSCP) system. We designed three probe sets for genes specific to Gram-positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram-positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain-specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target-specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug-resistant Gram-positive pathogens from sepsis patients' blood.

Effects of pelvic organ prolapse ring pessary therapy on intravaginal microbial flora.

The purpose of this paper is to report our evaluation of changes in intravaginal microbial flora after ring pessary therapy for pelvic organ prolapse (POP) using conventional and our clone library method. Thirteen patients with POP who were fitted with a ring pessary participated in this longitudinal study that incorporates data from before and 1 month after beginning ring pessary therapy. Changes in intravaginal microbial flora were evaluated by conventional methods, i.e., vaginal pH, lactobacillary grade (LAC grade), Nugent score, and culture-based bacterial detection methods. In addition, we performed our clone library method using 16S ribosomal RNA (rRNA) sequencing of vaginal fluid. Conventional methods revealed that most patients had abnormal intravaginal microbial flora. Mean numbers of detected bacterial species by the culture-based and our clone library method were 3.1 (1-6) and 11.8 (1-25), respectively. Our clone library method showed that Lactobacillus spp. increased in four and decreased in two cases after ring pessary therapy but reappeared after therapy in two cases; no Lactobacillus spp. were detected in five cases. Our study showed that ring pessary therapy did not always disturb intravaginal microbial flora, especially for patients with Lactobacillus spp. prior to ring pessary insertion. Anaerobic circumstances in the vagina after therapy seem to have induced the growth of anaerobic bacteria.

Individuality, phenotypic differentiation, dormancy and 'persistence' in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology.

For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically 'nonculturable' on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as 'persisters'. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one's bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.

Target-Enriched Multiplex PCR (Tem-PCR) Assay for Simultaneous Detection of S almonella spp., L isteria Monocytogenes and E scherichia Coli O157:H7 in Food

A novel target-enriched multiplex polymerase chain reaction (Tem-PCR) assay for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 was developed in the present study. The invA, hly and rfbE genes were selected as target genes for identifying Salmonella spp., L. monocytogenes and E. coli O157:H7, respectively. Three pairs of target-enriched long primers with a composition of pathogen-specific PCR primer combined with a universal oligonucleotide sequence at 5′ segment of each pathogen-specific PCR primer were designed. Following optimization of reaction conditions, the Tem-PCR assay for detecting Salmonella spp., L. monocytogenes and E. coli O157:H7 was successfully developed, which was able to simultaneously detect the three foodborne pathogens from pure cultures with an analytical detection limit of ca. <110 cfu/mL for each target. The Tem-PCR assay showed a high specificity to the target bacteria without specific priming observed in nontarget bacteria and also showed a potential diagnostic capability in practice. Compared with conventional multiplex PCR assay, the Tem-PCR assay does need not to optimize the concentration of each pair of primers, suggesting an efficient detection method for foodborne pathogens in laboratory diagnosis. Practical Applications Applying the target-enriched multiplex polymerase chain reaction assay to 355 food samples, including poultry meat, raw pork, raw milk, egg, sausage, raw beef, milk powder and frozen meat, revealed that 21 food samples were positive in this assay, in accordance with the testing results of conventional culture-based method, indicating a rapid and reliable tool for efficient screening of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 from food.

Individuality, phenotypic differentiation, dormancy and 'persistence' in culturable bacterial systems: commonalities shared by environmental, laboratory, and clinical microbiology.

For bacteria, replication mainly involves growth by binary fission. However, in a very great many natural environments there are examples of phenotypically dormant, non-growing cells that do not replicate immediately and that are phenotypically 'nonculturable' on media that normally admit their growth. They thereby evade detection by conventional culture-based methods. Such dormant cells may also be observed in laboratory cultures and in clinical microbiology. They are usually more tolerant to stresses such as antibiotics, and in clinical microbiology they are typically referred to as 'persisters'. Bacterial cultures necessarily share a great deal of relatedness, and inclusive fitness theory implies that there are conceptual evolutionary advantages in trading a variation in growth rate against its mean, equivalent to hedging one's bets. There is much evidence that bacteria exploit this strategy widely. We here bring together data that show the commonality of these phenomena across environmental, laboratory and clinical microbiology. Considerable evidence, using methods similar to those common in environmental microbiology, now suggests that many supposedly non-communicable, chronic and inflammatory diseases are exacerbated (if not indeed largely caused) by the presence of dormant or persistent bacteria (the ability of whose components to cause inflammation is well known). This dormancy (and resuscitation therefrom) often reflects the extent of the availability of free iron. Together, these phenomena can provide a ready explanation for the continuing inflammation common to such chronic diseases and its correlation with iron dysregulation. This implies that measures designed to assess and to inhibit or remove such organisms (or their access to iron) might be of much therapeutic benefit.

Comparative Study of Real Time Quantitative PCR for vanA Gene with the Conventional Culture - Based Method for the Detection of Vancomycin - Resistant Enterococci

Background: Vancomycin-resistant enterococci (VRE) are considered third leading cause of nosocomial infection. So, an effective, perfect and timely detection of VRE is required to start proper therapy and thus better patient outcome. Objectives: To compared the performance of real time quantitative PCR (Q-PCR) for vanA gene with the conventional culture-based method for the detection of VRE. Methodology: A total of 170 strains of enterococci; 95 vancomycin resistant strains and 75 vancomycin sensitive enterococci (VSE) isolated from different clinical specimens were detected by conventional culture based assay, at the Bacteriology Lab of King Fahd Hospital in Al- Madinah Al-Monawarah, KSA. All the strains were further tested for Van A gene by Q- PCR. Results: sensitivity and specificity of Q-PCR for VanA genotype were found to be 100% and 98.66% respectively. Conclusions: Q-PCR is a rapid and accurate technique to detect vanA in enterococci, thus it could be useful to settle on the treatment modalities of infections caused by VRE

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.

Evaluating the toxic effect of an antimicrobial agent on single bacterial cells with optical tweezers.

We implement an optical tweezers technique to assess the effects of chemical agents on single bacterial cells. As a proof of principle, the viability of a trapped Escherichia coli bacterium is determined by monitoring its flagellar motility in the presence of varying concentrations of ethyl alcohol. We show that the "killing time" of the bacterium can be effectively identified from the correlation statistics of the positional time series recorded from the trap, while direct quantification from the time series or associated power spectra is intractable. Our results, which minimize the lethal effects of bacterial photodamage, are consistent with previous reports of ethanol toxicity that used conventional culture-based methods. This approach can be adapted to study other pairwise combinations of drugs and motile bacteria, especially to measure the response times of single cells with better precision.

Development of a multiplex PCR assay for rapid and simultaneous detection of four genera of fish pathogenic bacteria.

Species of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus are the most common fish pathogenic bacteria that cause economically devastating losses in aquaculture. A multiplex polymerase chain reaction (mPCR) was developed for the simultaneous detection and differentiation of the four genera of fish pathogenic bacteria. Through the use of genus-specific primers instead of species-specific ones, the current mPCR covered much more target bacterial species compared with previously reported species-specific mPCR methods. The specificity of the four putative genus-specific primers was validated experimentally while used exclusively (uniplex PCR) or combined (mPCR) against bacterial genomic DNA templates of the target bacteria and nontarget bacteria. The PCR amplicons for the following genera were obtained as expected: Aeromonas (875bp), Vibrio (524bp), Edwardsiella (302bp) and Streptococcus (197bp), and the fragments could be separated clearly on the agarose gel electrophoresis. The mPCR did not produce nonspecific amplification products when used to amplify 21 nontarget species of bacteria. The mPCR detection limits for each target bacterial genera were 50 colony-forming units (CFU) in pure culture and 100CFU in fish tissue samples. In conclusion, the mPCR assay was proven to be a powerful alternative to the conventional culture-based method, given its rapid, specific, sensitive and reliable detection of target pathogens. The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus-specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species-specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This mPCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice.

Simultaneous Detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus by Multiplex Real-Time PCR Assays Using High-Resolution Melting

A method combining multiplex real-time polymerase chain reaction (PCR) with high-resolution melting (HRM) analysis for rapid and specific simultaneous detection of Salmonella, Listeria monocytogenes, and Staphylococcus aureus was developed. The method included a melting-curve analysis of products and was evaluated by specificity, sensitivity and reproducibility analyses. Sensitivity and reproducibility analyses was both conducted by genomic DNA extracted from serial dilutions for each target pathogen. Assays with artificially inoculated and naturally contaminated samples after enrichment were also conducted. In the specificity test, there was no nonspecific amplification of the 44 nontarget pathogens, whereas the actual Tm values were 79.38 ± 0.14, 82.54 ± 0.15, and 77.36 ± 0.14 °C for Salmonella, L. monocytogenes, and S. aureus, respectively. The sensitivity of the method was 3.5 × 102 CFU ml−1 for Salmonella and L. monocytogenes and 3.5 × 103 CFU ml−1 for S. aureus. The coefficients of variation of Tm values ranged 0.51–1.03 % for Salmonella, 1.63–2.11 % for L. monocytogenes, and 0.75–2.17 % for S. aureus in intraassay, and ranged 0.81–2.43 % for Salmonella, 1.97–2.35 % for L. monocytogenes, and 0.93–3.93 % for S. aureus in interassay. The detection limit in artificially inoculated samples (n = 50) was 5 CFU (25 g)−1 food for the three tested pathogens. In the naturally contaminated samples (n = 120),Salmonella DNA was detected by HRM, sequencing, and conventional culture-based methods at a positive rate of 25.00, 25.00, and 24.17 %, respectively; the corresponding rates for L. monocytogenes were 14.17, 14.17, and 14.17 %, respectively, while those for S. aureus were 16.7, 16.7, and 16.7 %, respectively.

Bacterial diversity in the indoor air of pharmaceutical environment

To monitor bacterial diversity of ISO Class 8 pharmaceutical clean room environment using conventional culture-based methods and pyrosequencing analysis. Bacterial isolates were obtained through viable particulate air monitoring, passive air monitoring and surface-monitoring procedures. A total of 157 bacterial isolates were obtained and assigned to four different phyla, Actinobacteria, Firmicutes, Proteobacteria and Deinococcus-Thermus, encompassing 52 species of 24 genera based on 16S rRNA gene sequence analysis. The genera Micrococcus and Staphylococcus were found as the main bacterial groups among the isolates. However, a big discrepancy was found between the culture based and pyrosequencing results. A total of 11409 quality reads were obtained from the pyrosequencing analysis, and the subsequent phylogenetic analysis indicated that Proteobacteria was the most abundant group at phylum level, followed by Actinobacteria and Firmicutes. Bacillus, Propionibacterium and Acinetobacter were identified as the most abundant genera by the pyrosequencing analysis. The culture-based results were in line with previous reports on the airborne bacterial composition of various environments, but the pyrosequencing analysis revealed a unique diversity of bacteria in this case. No significant pathogens above Riskgroup 2 were found from either culture based or pyrosequencing studies. The presence of various bacterial taxa including a number of groups, whose presence in air is previously unknown, was confirmed through this analysis. The main source of bacteria in the indoor air environment of pharmaceutical processes is likely human, but no significant primary pathogens were detected. Culture-based analysis may give limited information on the bacterial diversity of air environment.