Evaluation of the InTray and Compact Dry culture systems for the diagnosis of urinary tract infections in patients presenting to primary health clinics in Harare, Zimbabwe.

Antimicrobial resistance surveillance data is lacking from many resource-limited settings mainly due to limited laboratory testing. Novel culture systems may address some of the limitations of conventional culture media and expand the availability of microbiology services. The aims of this study were to evaluate the performance of InTray COLOREX Screen/ESBL and Compact Dry for the detection of uropathogens and of extended-spectrum beta-lactamase (ESBL)-producing organisms from urine samples. Urines samples were collected from patients presenting with symptoms of urinary tract infection to primary care clinics in Harare. Performance of the InTray COLOREX Screen, ESBL and Compact Dry chromogenic media were compared to the reference of culture using Brilliance UTI agar and conventional antimicrobial susceptibility testing. A total of 414 samples were included in the analysis. Of the included samples, 98 were positive on Brilliance UTI agar and 83 grew Enterobacterales. The sensitivities and specificities for Enterobacterales were 89.2% (95% CI 80.4-94.9) and 98.2% (95% CI 96.1-99.3) for InTray Screen and 95.2% (95% CI 88.1-98.7) and 99.7% (95% CI 98.3-100) for Compact Dry. Extended-spectrum beta-lactamases were present in 22 isolates from the Brilliance UTI agar. The sensitivity of the InTray COLOREX ESBL culture plates for the detection of ESBL-producing organisms was 95.5% (95% CI 77.2-99.9) and specificity was 99.5% (95% CI 98.2-99.9%). Our findings show good performance of the novel culture systems for the detection of uropathogens and ESBL-producing organisms. Both systems have several advantages over conventional media and have the potential to expand and decentralize laboratory testing.

Comparative study of Legiolert with ISO 11731-1998 standard method-conclusions from a Public Health Laboratory.

BACKGROUND: Legionella pneumophila (L. pneumophila) is responsible for 96% of Legionnaires' disease (LD) and 10% of all worldwide pneumonia cases. Legiolert™, a liquid culture method for most probable number (MPN) enumeration of L. pneumophila, was developed by IDEXX Laboratories. The method detects all serogroups of L. pneumophila in potable and non-potable water samples. OBJECTIVE: The goal of this study is to establish that Legiolert is a suitable alternative method to meet testing requirements in Spain for the enumeration of Legionella in water samples. METHODOLOGY: The laboratory analyzed 118 environmental water samples from the Barcelona region (56 potable and 62 non-potable) in parallel by the Standard method for detection and enumeration of Legionella (ISO 11731:1998) and by Legiolert. Comparison of the recovery of the alternative method (Legiolert) and the Standard was made using ISO 17994:2014 and McNemar's binomial test statistical methods. RESULTS: 44 samples were positive for Legionella (36 potable and 8 non-potable). Legiolert and the Standard method detected a similar percentage of positive samples, with Legiolert being slightly higher (31 vs 30%) and detecting higher concentrations of Legionella within the samples. ISO 17994:2014 analysis of the potable water samples found Legiolert was more sensitive than the Standard at detecting Legionella, even when complete Legionella species (L. spp.) results were considered for both methods. The two methods also demonstrated equivalent detection of L. spp. according to the McNemar's test. The comparison is significantly more in favor of Legiolert when only L. pneumophila results are considered. Each confirmation run with material extracted from positive Legiolert wells contained L. pneumophila, giving the method a specificity of 100%. Although statistical results for non-potable waters are not included because of the low number of samples, the two methods trended towards equivalence. CONCLUSIONS: Relative to the Standard method, Legiolert has a greater sensitivity and selectivity, and appears to have higher recovery for L. pneumophila, and equivalent recovery when L. spp. is included in the comparison. Legiolert also has high specificity. The procedural advantages of Legiolert allow laboratories to save on resources, costs, and time and consequently to test more frequently. In conclusion, the study finds IDEXX Legiolert a suitable alternative to ISO 11731:1998.

Incubation temperature and culture medium formulation impact the accuracy of pour-plate techniques for the enumeration of industrial Bacillus assemblages.

Aerobic plate counting assays based on the pour-plate technique are frequently used to enumerate microbial products; however, colony swarming and merging at the agar surface can reduce the accuracy of these assays. Some plating methods mitigate this risk through the inclusion of strategies including agar overlays; however, these interventions may be inadequate to mitigate swarming and merging of certain Bacillus colonies. In the present study, we assessed the accuracy of several pour-plate techniques for the enumeration of a mixed-species Bacillus assemblage. Tested modifications included a customized culture medium formulation, agar overlays, decreased incubation times and increased incubation temperature. Methods which produced countable plates were assessed for agreement with a Bacillus-specific plate counting assay and with total cell counts rendered by flow cytometry. While all tested pour-plate methods underestimated Bacillus endospore concentrations relative to flow cytometry and customized spread-plating, our results suggest that increasing incubation temperature and the inclusion of bile salts into culture medium formulations can improve the accuracy of pour-plate techniques when used to enumerate Bacillus assemblages by decreasing the incidence of spreading colonies. As Bacillus endospore preparations become more ubiquitous in the market, familiar enumeration methods such as the pour-plate technique may require methodological modifications to ensure that the cGMP compliance of Bacillus-based microbial products is assessed accurately.

Improvement of the detection efficiency of 3M™ molecular detection system for Campylobacter in poultry using nitrogen-doped carbon nanodots.

This study was performed to examine whether the use of nitrogen-doped carbon nanodots (N-CNDs) can improve the detection sensitivity of the 3 M™ molecular detection system (MDS) for Campylobacter. N-CNDs were added to a Campylobacter enrichment broth (CEB) at concentrations of 5 and 10 mg/mL (NCEB-5 and NCEB-10, respectively). Campylobacter coli, C. jejuni, and C. lari were inoculated into the broths. The broth cultures were then irradiated with light-emitting diode (LED) at 425 nm for 1 h and incubated at 42 °C for 6 h, and then grown on modified charcoal cefoperazone deoxycholate agar (mCCDA). The detection rates of the MDS and a conventional method (plating an enriched sample on mCCDA and analyzing a colony on mCCDA with PCR) for Campylobacter in chicken and duck carcasses were compared. The detection rates from the MDS were compared after enrichment in CEB and NCEB-5 at 3, 5, 6, 7, 9, 12, and 24 h. When 5 mg/mL of N-CNDs was added to the CEB followed by irradiation at 425 nm, growth of the Campylobacter was accelerated. In addition, the qualitative test was more sensitive in the MDS than in the conventional method, and the detection time was shortened in CEB enriched with N-CNDs. These results indicate that adding N-CNDs to CEB can improve the detection efficiency of MDS.

A high-throughput ultrasonic spraying inoculation method promotes colony cultivation of rare microbial species.

Current method for obtaining microbial colonies still relies on traditional dilution and spreading plate (DSP) procedures, which is labor-intensive, skill-dependent, low-throughput and inevitably causing dilution-to-extinction of rare microorganisms. Herein, we proposed a novel ultrasonic spraying inoculation (USI) method that disperses microbial suspensions into millions of aerosols containing single cells, which lately be deposited freely on a gel plate to achieve high-throughput culturing of colonies. Compared with DSP, USI significantly increased both distributing uniformity and throughput of the colonies on agar plates, improving the minimal colony-forming abundance of rare Escherichia coli mixed in a lake sample from 1% to 0.01%. Applying this novel USI to a lake sample, 16 cellulose-degrading colonies were screened out among 4766 colonies on an enlarged 150-mm-diameter LB plate. Meanwhile, they could only be occasionally observed when using commonly used DSP procedures. 16S rRNA sequencing further showed that USI increased colony-forming species from 11 (by DSP) to 23, including seven completely undetectable microorganisms in DSP-reared communities. In addition to avoidance of dilution-to-extinction, operation-friendly USI efficiently inoculated microbial samples on the agar plate in a high-throughput and single-cell form, which eliminated masking or out-competition from other species in associated groups, thereby improving rare species cultivability.

Experimental setup and image processing method for automatic enumeration of bacterial colonies on agar plates.

Automated colony counting methods have long been known in Microbiology. Numerous methods for automated image analysis have been described and a wide range of commercial products exists. Known advantages are saving cost by reducing enumeration time, automatic documentation, reproducibility, and operator independence. Still, even today the realization of all advantages of automated image analysis makes it necessary to either invest in an expensive, high performance commercial system, or to acquire expert knowledge in image processing. This is a considerable obstacle for many laboratories, and the reason why manual colony counting is still done frequently. This article describes an easy to apply automatic colony counting system-including suggestions for sample preparation-that can be put into operation with basic knowledge of image processing and low budget.

Pyphe, a python toolbox for assessing microbial growth and cell viability in high-throughput colony screens.

Microbial fitness screens are a key technique in functional genomics. We present an all-in-one solution, pyphe, for automating and improving data analysis pipelines associated with large-scale fitness screens, including image acquisition and quantification, data normalisation, and statistical analysis. Pyphe is versatile and processes fitness data from colony sizes, viability scores from phloxine B staining or colony growth curves, all obtained with inexpensive transilluminating flatbed scanners. We apply pyphe to show that the fitness information contained in late endpoint measurements of colony sizes is similar to maximum growth slopes from time series. We phenotype gene-deletion strains of fission yeast in 59,350 individual fitness assays in 70 conditions, revealing that colony size and viability provide complementary, independent information. Viability scores obtained from quantifying the redness of phloxine-stained colonies accurately reflect the fraction of live cells within colonies. Pyphe is user-friendly, open-source and fully documented, illustrated by applications to diverse fitness analysis scenarios.

Computer Vision Approach for the Determination of Microbial Concentration and Growth Kinetics Using a Low Cost Sensor System.

The measurement of microbial contamination is of primary importance in different fields, from environmental monitoring to food safety and clinical analysis. Today, almost all microbiology laboratories make microbial concentration measurements using the standard Plate Count Technique (PCT), a manual method that must be performed by trained personnel. Since manual PCT analysis can result in eye fatigue and errors, in particular when hundreds of samples are processed every day, automatic colony counters have been built and are commercially available. While quick and reliable, these instruments are generally expensive, thus, portable colony counters based on smartphones have been developed and are of low cost but also not accurate as the commercial benchtop instruments. In this paper, a novel computer vision sensor system is presented that can measure the microbial concentration of a sample under test and also estimate the microbial growth kinetics by monitoring the colonies grown on a Petri dish at regular time intervals. The proposed method has been in-house validated by performing PCT analysis in parallel under the same conditions and using these results as a reference. All the measurements have been carried out in a laboratory using benchtop instruments, however, such a system can also be realized as an embedded sensor system to be deployed for microbial analysis outside a laboratory environment.

Automated image analysis with ImageJ of yeast colony forming units from cannabis flowers.

Currently, in the state of Colorado and all other states within the United States of America with legalized marijuana programs, testing is required for bacteria, yeast, and mold on marijuana products. The Code of Colorado Regulations, 1 CCR 212-1, considers a passing result when a 1 g sample contains <104 colony forming units (CFU) for the total yeast and mold count (TYMC). These measurements are usually obtained by manually counting colonies on petri-dishes or 3 M™ Petrifilms™, which is a time consuming and user subjective process. Therefore, an automated counting method utilizing ImageJ has been developed for CFU analysis of TYMC on Petrifilms. The performance of this colony counting method was demonstrated by comparing manual and automated counts from marijuana flower samples containing spikes of Candida albicans as well as samples that tested positive for the presence of yeast and mold. Fifteen images of Petrifilms showing various concentrations of colonies were studied by fifteen users at two institutions using both the automated and manual counting methods. All counts from the automated ImageJ procedure were within 12% of those obtained manually. In twelve out of fifteen Petrifilms, the average count of the automated method was statistically similar to the manual counts. The statistical differences of the other three samples were observed to be random and caused by user errors. The automated counting method could be used to quickly count numbers that are as high as 400 CFUs, reducing time of analysis with improved documentation because the images and the electronic colony counts can be saved on a computer or cloud for long term storage and data access.

HiCrome Bacillus agar for presumptive identification of Bacillus and related species isolated from honey samples.

This study aimed to evaluate the performance of Hicrome Bacillus™ agar for isolation and rapid identification of the aerobic spore-forming bacteria most frequently found in honey samples. A collection of 197 bacterial isolates of Bacillus, Brevibacillus, Lysinibacillus, Paenibacillus, and Rummeliibacillus belonging to different species that have been reported in honey were screened for their abilities to grow and for their colony colors and medium appearance in HiCrome Bacillus agar. Also, 21 strains from culture collections were used for comparison and quality controls. A flowchart utilizing a combination of colony and media characteristics in the chromogenic medium and a set of simple biochemical and morphological tests were elaborated for quick presumptive identification. A procedure for direct isolation from honey samples was developed. In conclusion, HiCrome Bacillus agar in combination with simple microbiological tests was highly useful for rapid and reliable identification of most Bacillus, Brevibacillus, Lysinibacillus and Paenibacillus species commonly found in honey samples facilitating isolation from polymicrobial honey.

Assessment of two selective agar media to isolate colistin-resistant bovine Escherichia coli: Correlation with minimal inhibitory concentration and presence of mcr genes.

The identification of colistin-resistant enterobacteria in veterinary medicine is impaired by the absence of first-line reliable phenotypic assay. The purpose of this study was to assess two selective agar media for the detection of colistin-resistant bovine pathogenic Escherichia coli. A total of 158 E. coli (46 R , 96 I and 16 S at the disk diffusion assay) isolated between 2013 and 2018 from <3 month-old calves suffering enteritis or septicaemia, were (i) tested by the broth dilution assay to determine colistin Minimal Inhibitory Concentrations (MIC); (ii) streaked on CHROMID® Colistin_R and CHROMagar™ COL-APSE agar plates; (iii) submitted to a pentaplex PCR to identify the presence of mcr-1 to mcr-5 genes. Of the 92 E. coli growing on both agar media, 90 had a MIC > 2.0 µg/ml as had the 3 E. coli that grew only on the CHROMID® Colistin_R agar medium and one E. coli that grew on neither agar media. Therefore, the positive predictive values of the CHROMID® Colistin_R and CHROMagar™ COL-APSE agar media were both 0.98 whereas their negative predictive values were 0.98 and 0.94, respectively. Also noteworthy 43 of the 46 R isolates had a MIC > 2.0 µg/ml and grew on both selective media as did half of the 96 I isolates and only 1 of the S isolates. Conversely, only 30 of the 90 isolates that grew on both agar media and with a MIC > 2.0 µg/ml tested positive for the mcr-1 or mcr-2 genes with the pentaplex PCR. These two selective agar media can be used to reliably detect colistin-resistant E. coli. Positive growth was highly correlated with R results at the disk diffusion assay, but not with the presence of mcr genes.

Performance of yeast-like cell counting (YLCC) using the Sysmex UF-1000i for clinical candiduria screening.

Candiduria is common in clinical practice. However, an effective and convenient assay to screen for candiduria is still needed. This study aimed to evaluate the performance of the Sysmex UF-1000i urine analyzer for yeast-like cell counting (YLCC) to screen for candiduria prior to urine culture. We retrospectively analyzed data from 5233 urine samples from 1813 patients, including 837 males and 976 females. Urine culture and urinalysis-obtained YLCC data were used to estimate the performance of YLCC in diagnosing candiduria. Different cutoff values were used to calculate sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The YLCC-positive rates differed according to the Candida colony-forming units (CFU) counts in the urine samples. A sharp drop in YLCC-positive rate (from 64.3 to 22.0%) was observed between the urine groups with 104 CFUs and 103 CFUs. A cutoff value of 0 YLCs/µL results in the highest Youden index (0.71) with 77.04% sensitivity and 93.68% specificity. In a group of 34 hospitalized candiduria patients with serial urinalysis data, 25 were YLCC-positive before urine culture. In conclusion, YLCC with the Sysmax UF-1000i could serve as an auxiliary technique to exclude culture-negative specimens prior to urine culture. Positive YLCC results could imply candiduria, especially when persistent YLCC-positive results were observed.

Measuring Antibacterial Autophagy.

Bacteria that escape from membrane-enclosed vacuoles to the cytosol of cells are targeted by autophagy, which recognizes and captures bacteria into autophagosomes wherein their proliferation is restricted. Here we discuss two means by which antibacterial autophagy is assessed: (1) the visualization and enumeration of autophagy protein recruitment to the vicinity of cytosolic bacteria by means of immunofluorescence microscopy and (2) the measurement of autophagy-dependent restriction of bacterial proliferation by means of colony-forming unit assay.

Recipes and Tools for Culture of Escherichia coli.

In this article, we provide information about culture media, including minimal liquid media, rich liquid media, solid media, top agar, and stab agar. We also provide descriptions and useful information about tools used with growth media such as inoculating loops, sterile toothpicks, and spreaders. © 2018 by John Wiley & Sons, Inc.

Growth of E. coli in Liquid Medium.

We describe the procedure for inoculating overnight (starter) cultures of E. coli from a single colony, along with considerations for growing larger cultures. We also include two methods for monitoring the number of cells per unit volume (density) of liquid cultures using a spectrophotometer and a hemacytometer or "count slide." © 2018 by John Wiley & Sons, Inc.

Noise-free microbial colony counting method based on hyperspectral features of agar plates.

A noise-free bacterial colony counting method identifying noise (i.e., sausage, bacon, and millet fragments) with similar colors or shapes to those of colonies was developed for food quality assessment. First, spectral features corresponding to colony cluster regions and background regions (agar medium and food fragments) were extracted after collection of hyperspectral images. A cluster-segmenting calibration model that could identify colony clusters and background regions was developed. Second, spectral features of colony centers and borders were extracted, and a colony-separating calibration model that could separate single colonies from clusters (multiple colonies contacting each other) was developed. Third, each pixel of an agar plate hyperspectral image was identified using established calibration models, enabling the colonies on the agar plate to be counted successfully (R2 = 0.9998). The results demonstrated that the proposed method could identify the noises caused by food fragments with similar colors or shapes to those of colonies.

Anabaena sp. strain PCC 7120: Laboratory Maintenance, Cultivation, and Heterocyst Induction.

Anabaena sp. strain PCC 7120 is a multicellular, filamentous, freshwater cyanobacterium that is capable of differentiating specialized heterocyst cells for nitrogen fixation. This unit includes protocols for the growth and maintenance of Anabaena appropriate for a research or teaching laboratory. Controlled induction and assessment of heterocyst development is also covered. © 2018 by John Wiley & Sons, Inc.

Are bacterial culture quantifications reliable? Comparative performance of the WASP automated inoculation instrument in the era of ISO 15189 accreditation.

PURPOSE: Isolating colonies and obtaining accurate colony counts from bacterial cultures are critical steps for the optimal management of infected patients. The uncertainties in the colony count results from the bacterial cultures were evaluated by verifying the performance of the WASP inoculation system according to the International Organization for Standardization (ISO) 15189 standard. METHODOLOGY: We first (i) evaluated the cross-contamination and precision of the WASP instrument (Copan Diagnostics, Italy) and (ii) established enumeration reading grids for urine, swab, bronchopulmonary specimens (BPSs) and catheter tip cultures. Subsequently, 72 clinical samples were tested to compare the results of the WASP, PREVI Isola (bioMérieux, France) and manual inoculation methods. RESULTS: The WASP method did not show cross-contamination. The coefficient of variation for the colony counts in the repeatability experiment was evaluated for 10 µl and 30 µl loop protocols and determined to be 29 and 14 %, respectively. The agreement between the automated and manual methods and between the automated methods for the colony counts was high (94.4 and 100 %, respectively). The WASP method yielded better isolation quality compared to the manual method (P=0.020) and to the PREVI Isola only when polymicrobial specimens were considered (P=0.014). For quantification evaluation, the measurement uncertainty was evaluated to 1.8×103 c.f.u. ml-1 for a suspension of Escherichia coli at 104 c.f.u. ml-1. CONCLUSION: We report the verification of the performance of the WASP instrument and describe a rapid procedure for achieving semi-quantitative cultures from BPSs and catheter tips. Quantitative interpretation of the bacterial cultures should be performed with caution.

Automated counting of bacterial colonies on agar plates based on images captured at near-infrared light.

Counting colonies is usually used in microbiological analysis to assess if samples meet microbiological criteria. Although manual counting remains gold standard, the process is subjective, tedious, and time-consuming. Some developed automatic counting methods could save labors and time, but their results are easily affected by uneven illumination and reflection of visible light. To offer a method which counts colonies automatically and is robust to light, we constructed a convenient and cost-effective system to obtain images of colonies at near-infrared light, and proposed an automatic method to detect and count colonies by processing images. The colonies cultured by using raw cows' milk were used as identification objects. The developed system mainly consisted of a visible/near-infrared camera and a circular near-infrared illuminator. The automatic method proposed to count colonies includes four steps, i.e., eliminating noises outside agar plate, removing plate rim and wall, identifying and separating clustered or overlapped colonies, and counting colonies by using connected region labelling, distance transform, and watershed algorithms, etc. A user-friendly graphic user interface was also developed for the proposed method. The relative error and counting time of the automatic counting method were compared with those of manual counting. The results showed that the relative error of the automatic counting method was -7.4%~ + 8.3%, with average relative error of 0.2%, and the time used for counting colonies on each agar plate was 11-21 s, which was 15-75% of the time used in manual counting, depending on the numbers of colonies on agar plates. The proposed system and automatic counting method demonstrate promising performance in terms of precision, and they are robust and efficient in terms of labor- and time-savings.

Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media.

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.