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.

Combined selective culture and molecular methods for the detection of carbapenem-resistant organisms from fecal specimens.

Detection of patients with intestinal colonization of carbapenem-resistant organisms (CRO), or more specifically carbapenemase-producing (CP) CRO, can prevent their transmission in healthcare facilities and aid with outbreak investigations. The objective of this work was to further develop and compare methods that combine selective culture and/or PCR to rapidly detect and recover CRO from fecal specimens. Molecularly characterized Gram-negative bacilli (n = 62) were used to spike fecal samples to establish limit of detection (LOD; n = 12), sensitivity (n = 28), and specificity (n= 21) for 3 methods to detect CP-CRO: direct MacConkey (MAC) plate and Xpert Carba-R (Cepheid) on growth, MAC broth and Carba-R testing of the broth, and direct testing by Carba-R. This was followed by a clinical study comparing methods in parallel for 286 fecal specimens. The LOD ranged from 102-105 CFU/mL depending on the carbapenemase gene and method. Combined culture/PCR methods had a sensitivity of 100%, whereas direct Carba-R testing had a sensitivity of 96% for the detection of CP-CRO. All methods had specificities of 100%. The prevalence of CP-CRO (0.7%) and non-CP-CRO (5.2 %) were low in the clinical study, where all methods demonstrated 100% agreement. The three methods performed comparably in detecting CP-CRO. Direct Carba-R testing had a higher LOD than the combined selective culture methods, but this may be offset by its rapid turnaround time for detection of CP-CRO. The selective culture methods provide the benefit of simultaneously isolating CP-CRO in culture for follow-up testing and detecting non-CP-CRO.

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.

Determination of available breaking stress of agar and gellan gum plate culture methods and the duration of bacterial culture under strong acidic conditions.

AIMS: Several acidophilic bacteria have not been cultured, primarily owing to the lack of suitable culture methods under strong acidic conditions. This study aimed to quantitatively evaluate the strengths of the agar plates (AP) and gellan gum plates (GP), and optimal culture periods under strong acidic conditions. METHODS AND RESULTS: To define the lower limit of plate strength for bacterial isolation culture, the diameter of Escherichia coli K12 colonies and the breaking stress of plates at different concentrations of gelling agents, medium composition and pH conditions were determined. The lower limit of available strength of AP and GP was 19·6 and 14·8 kPa, respectively. Medium composition slightly affected AP breaking stress, although GP with a high cationic concentration medium could not be prepared. CONCLUSIONS: Assessment of the strength limits of AP and GP revealed that AP is not suitable for prolonged bacterial culture (≥72 h). Furthermore, GP was completely ineffective for bacterial culture under highly acidic conditions (≤pH 1·0). SIGNIFICANCE AND IMPACT OF THE STUDY: Our quantitative evaluation method based on breaking stress is a potentially valuable tool to understand the state and the suitable limit of plate culture methods in more detail under various conditions.

Identification of novel monoclonal antibodies targeting the outer membrane protein C and lipopolysaccharides for Escherichia coli O157:H7 detection.

AIMS: To identify and evaluate the application of two novel monoclonal antibody (mAb) 2G12 against outer membrane protein (Omp) C and mAb 12B1 targeting the O chain of the lipopolysaccharides (LPS) of Escherichia coli O157:H7 (ECO157). METHODS AND RESULTS: The sensitivity and specificity of these two antibodies were evaluated with eight ECO157 strains and 68 untargeted strains. mAb 2G12 and 12B1 had no detectable binding with any of the non-O157 strains at 6·0 log10 CFU per ml, while its high specificity and affinity remained with all ECO157 strains. When a higher level (8·0 log10 CFU per ml) was tested, 2G12 and 12B1 did not react with 82·35 and 97·06% of the non-O157 strains respectively. Based on the pair of two antibodies, the sandwich enzyme-linked immunosorbent assay detected 100% (8/8) of ECO157 strains and none of the non-ECO157 strains. The detection limit of ECO157 strains in pure culture were 4·2 ± 0·2 log10 CFU per ml. When the developed test was applied to artificially inoculated beef samples, the detection limit was 6·0 log10 CFU per gram without enrichment and 1·0 log10 CFU per gram after 12 h of enrichment. CONCLUSIONS: The two novel antibodies identified in this study served as great candidates for the recovery, and detection of ECO157 from different environmental and food samples. SIGNIFICANCE AND IMPACT OF THE STUDY: ECO157-specific detection was improved by a combination of the novel OmpC mAb and LPS mAb with defined target antigen and good specificity.

Rapid culture-based identification of Shiga toxin-producing Escherichia coli and Shigella spp./Enteroinvasive E. coli using the eazyplex® EHEC complete assay.

Shiga toxin-producing Escherichia coli (STEC) and Shigella spp./enteroinvasive E. coli (EIEC) are common diarrheagenic bacteria that cause sporadic diseases and outbreaks. Clinical manifestations vary from mild symptoms to severe complications. For microbiological diagnosis, culture confirmation of a positive stool screening PCR test is challenging because of time-consuming methods for isolation of strains, wide variety of STEC pathotypes, and increased emergence of non-classical strains with unusual serotypes. Therefore, molecular assays for the rapid identification of suspect colonies growing on selective media are very useful. In this study, the performance of the newly introduced eazyplex® EHEC assay based on loop-mediated isothermal amplification (LAMP) was evaluated using 18 representative STEC and Shigella strains and 31 isolates or positive-enrichment broths that were collected from clinical stool samples following screening by BD MAX™ EBP PCR. Results were compared to real-time PCR as a reference standard. Overall, sensitivities and specificities of the eazyplex® EHEC were as follows: 94.7% and 100% for Shiga toxin 1 (stx1), 100% and 100% for stx2, 93.3% and 97.1% for intimin (eae), 100% and 100% for enterohemolysin A (ehlyA), and 100% and 100% for invasion-associated plasmid antigen H (ipaH) as Shigella spp./EIEC target, respectively. Sample preparation for LAMP took only some minutes, and the time to result of the assay ranged from 8.5 to 13 min. This study shows that eazyplex® EHEC is a very fast and easy to perform molecular assay that provides reliable results as a culture confirmation assay for the diagnosis of STEC and Shigella spp./EIEC infections.

Interventions to reduce contaminated aerosols produced during dental procedures for preventing infectious diseases.

BACKGROUND: Many dental procedures produce aerosols (droplets, droplet nuclei and splatter) that harbour various pathogenic micro-organisms and may pose a risk for the spread of infections between dentist and patient. The COVID-19 pandemic has led to greater concern about this risk. OBJECTIVES: To assess the effectiveness of methods used during dental treatment procedures to minimize aerosol production and reduce or neutralize contamination in aerosols. SEARCH METHODS: Cochrane Oral Health's Information Specialist searched the following databases on 17 September 2020: Cochrane Oral Health's Trials Register, the Cochrane Central Register of Controlled Trials (CENTRAL) (in the Cochrane Library, 2020, Issue 8), MEDLINE Ovid (from 1946); Embase Ovid (from 1980); the WHO COVID-19 Global literature on coronavirus disease; the US National Institutes of Health Trials Registry (ClinicalTrials.gov); and the Cochrane COVID-19 Study Register. We placed no restrictions on the language or date of publication. SELECTION CRITERIA: We included randomized controlled trials (RCTs) and controlled clinical trials (CCTs) on aerosol-generating procedures (AGPs) performed by dental healthcare providers that evaluated methods to reduce contaminated aerosols in dental clinics (excluding preprocedural mouthrinses). The primary outcomes were incidence of infection in dental staff or patients, and reduction in volume and level of contaminated aerosols in the operative environment. The secondary outcomes were cost, accessibility and feasibility. DATA COLLECTION AND ANALYSIS: Two review authors screened search results, extracted data from the included studies, assessed the risk of bias in the studies, and judged the certainty of the available evidence. We used mean differences (MDs) and 95% confidence intervals (CIs) as the effect estimate for continuous outcomes, and random-effects meta-analysis to combine data. We assessed heterogeneity. MAIN RESULTS: We included 16 studies with 425 participants aged 5 to 69 years. Eight studies had high risk of bias; eight had unclear risk of bias. No studies measured infection. All studies measured bacterial contamination using the surrogate outcome of colony-forming units (CFU). Two studies measured contamination per volume of air sampled at different distances from the patient's mouth, and 14 studies sampled particles on agar plates at specific distances from the patient's mouth. The results presented below should be interpreted with caution as the evidence is very low certainty due to heterogeneity, risk of bias, small sample sizes and wide confidence intervals. Moreover, we do not know the 'minimal clinically important difference' in CFU. High-volume evacuator Use of a high-volume evacuator (HVE) may reduce bacterial contamination in aerosols less than one foot (~ 30 cm) from a patient's mouth (MD -47.41, 95% CI -92.76 to -2.06; 3 RCTs, 122 participants (two studies had split-mouth design); very high heterogeneity I² = 95%), but not at longer distances (MD -1.00, -2.56 to 0.56; 1 RCT, 80 participants). One split-mouth RCT (six participants) found that HVE may not be more effective than conventional dental suction (saliva ejector or low-volume evacuator) at 40 cm (MD CFU -2.30, 95% CI -5.32 to 0.72) or 150 cm (MD -2.20, 95% CI -14.01 to 9.61). Dental isolation combination system One RCT (50 participants) found that there may be no difference in CFU between a combination system (Isolite) and a saliva ejector (low-volume evacuator) during AGPs (MD -0.31, 95% CI -0.82 to 0.20) or after AGPs (MD -0.35, -0.99 to 0.29). However, an 'n of 1' design study showed that the combination system may reduce CFU compared with rubber dam plus HVE (MD -125.20, 95% CI -174.02 to -76.38) or HVE (MD -109.30, 95% CI -153.01 to -65.59). Rubber dam One split-mouth RCT (10 participants) receiving dental treatment, found that there may be a reduction in CFU with rubber dam at one-metre (MD -16.20, 95% CI -19.36 to -13.04) and two-metre distance (MD -11.70, 95% CI -15.82 to -7.58). One RCT of 47 dental students found use of rubber dam may make no difference in CFU at the forehead (MD 0.98, 95% CI -0.73 to 2.70) and occipital region of the operator (MD 0.77, 95% CI -0.46 to 2.00). One split-mouth RCT (21 participants) found that rubber dam plus HVE may reduce CFU more than cotton roll plus HVE on the patient's chest (MD -251.00, 95% CI -267.95 to -234.05) and dental unit light (MD -12.70, 95% CI -12.85 to -12.55). Air cleaning systems One split-mouth CCT (two participants) used a local stand-alone air cleaning system (ACS), which may reduce aerosol contamination during cavity preparation (MD -66.70 CFU, 95% CI -120.15 to -13.25 per cubic metre) or ultrasonic scaling (MD -32.40, 95% CI - 51.55 to -13.25). Another CCT (50 participants) found that laminar flow in the dental clinic combined with a HEPA filter may reduce contamination approximately 76 cm from the floor (MD -483.56 CFU, 95% CI -550.02 to -417.10 per cubic feet per minute per patient) and 20 cm to 30 cm from the patient's mouth (MD -319.14 CFU, 95% CI - 385.60 to -252.68). Disinfectants ‒ antimicrobial coolants Two RCTs evaluated use of antimicrobial coolants during ultrasonic scaling. Compared with distilled water, coolant containing chlorhexidine (CHX), cinnamon extract coolant or povidone iodine may reduce CFU: CHX (MD -124.00, 95% CI -135.78 to -112.22; 20 participants), povidone iodine (MD -656.45, 95% CI -672.74 to -640.16; 40 participants), cinnamon (MD -644.55, 95% CI -668.70 to -620.40; 40 participants). CHX coolant may reduce CFU more than povidone iodine (MD -59.30, 95% CI -64.16 to -54.44; 20 participants), but not more than cinnamon extract (MD -11.90, 95% CI -35.88 to 12.08; 40 participants). AUTHORS' CONCLUSIONS: We found no studies that evaluated disease transmission via aerosols in a dental setting; and no evidence about viral contamination in aerosols. All of the included studies measured bacterial contamination using colony-forming units. There appeared to be some benefit from the interventions evaluated but the available evidence is very low certainty so we are unable to draw reliable conclusions. We did not find any studies on methods such as ventilation, ionization, ozonisation, UV light and fogging. Studies are needed that measure contamination in aerosols, size distribution of aerosols and infection transmission risk for respiratory diseases such as COVID-19 in dental patients and staff.

The best type of inoculum for testing the antifungal drug susceptibility of Microsporum canis: In vivo and in vitro results.

BACKGROUND: Data correlating in vitro drug susceptibility of Microsporum canis with clinical outcomes of its infections are lacking as well as the most suitable inoculum and incubation time in broth microdilution assays. OBJECTIVES AND METHODS: Microsporum canis strains were collected from animal hosts that tested positive (Group I; n = 13) and negative (Group II; n = 14) to this pathogen following itraconazole (ITC) therapy. In vitro ITC susceptibility was assessed according to the Clinical Laboratory Standards Institute (CLSI M38-A2) methodology using conidia, hypha-conidia and arthroconidia at 3 and 7 days of incubation in order to assess the most suitable inoculum and incubation time. Successively, ketoconazole (KTC), voriconazole (VRC), terbinafine (TRB), posaconazole (PSZ), fluconazole (FLC) and griseofulvin (GRI) susceptibilities were assessed using the chosen inoculum. RESULTS: The MIC values of ITC after three-day incubation were equal than those recorded after 7-day incubation. Itraconazole MICs were ≤1 µg/mL for strains from Group II and >1 µg/mL for those of Group II only when conidia were used. All strains showed high susceptibility to VRC, POS, TEB and low susceptibility to ITC, KTC, GRI and FLC regardless of the source and incubation time. CONCLUSIONS AND CLINICAL IMPORTANCE: Results suggest that correlation between the in vitro results and clinical outcome was observed only by incubating conidia for 3 days at 30 ± 2°C. These conditions might be most suitable to assess in vitro susceptibility of M. canis and assist in determining the occurrence of drug resistance and cross-resistance phenomena.

Comparison of predicted and impedance determined growth of Listeria innocua in complex food matrices.

Indirect impedance has been used for the detection and enumeration of bacteria, however there is limited data regarding the ability of the method to measure growth and inhibition of microorganisms in food in response to preservatives. The aim of this study was to evaluate the suitability of the technique to determine maximum growth rates of Listeria innocua (used as a surrogate for Listeria monocytogenes) in complex food matrices to which multiple preservative factors had been applied and assess the suitability of the data for use in predictive microbiology. Growth of L. innocua in laboratory medium (BHI broth) and two food matrices (zucchini purée and béarnaise sauce) under varying conditions of pH (5 & 5.3), water activity (0.93, 0.96 & 0.98) and acetic and propionic acid concentration (0, 1 & 2 mM) was monitored by the conductimetric Rapid Automated Bacterial Impedance Technology (R.A.B.I.T) system by means of CO2 emission for up to 120 h. Growth rates of L. innocua were determined for several conditions across the three test matrices and a good correlation between detection times and initial inoculum level was observed in most cases (R2 ≥ 0.82). However, growth of L. innocua was not detected in a large number of conditions and comparison of growth rates determined by indirect impedance to those determined by plate counts indicated that in general, the R.A.B.I.T. system under-estimated growth. This study demonstrates that there are limitations associated with the technology, and as a result the system may be unsuitable for measuring microbial growth rates in complex food matrices under the environmental conditions tested and within the time duration of the study.

Dwindling Utilization of Central Venous Catheter Tip Cultures: An Analysis of Sampling Trends and Clinical Utility at 128 US Hospitals, 2009-2014.

At 128 US hospitals, from 2009-2014, a 17% decline occurred annually in central venous catheter tips sent for culture: a 6-fold decrease from blood culture sampling trends. The positive predictive value was low (23%). Tip culture use often does not conform to recommendations and offers limited independent treatment opportunities.

Isothermal microcalorimetry for thermal viable count of microorganisms in pure cultures and stabilized formulations.

BACKGROUND: Quantification of viable microorganisms is an important step in microbiological research as well as in microbial product formulation to develop biological control products or probiotics. Often, the efficiency of the resulting product is dependent on the microbial cell density and their viability, which may decrease over time. Commonly, the number of viable cells is determined by serial dilution and plating techniques or flow cytometry. In 2017, we developed a mathematical model for isothermal microcalorimetry (IMC) data analysis and showed that the new method allows for a more rapid quantification of viable fresh and freeze-dried anaerobic Lactobacillus reuteri cells than traditional viable count methods. RESULTS: This study developed the new method further by applying it to well-known aerophilic plant-beneficial microbial species (Pseudomonas brassicacearum, Bacillus amyloliquefaciens subsp. plantarum and Clonostachys rosea) used in biological control products. We utilized IMC to quantify viable cells in microbial pure cultures as well as when coated onto wheat seeds. The results from this study confirmed that thermal viable count methods are more rapid and sensitive than traditional viable count techniques. Most interestingly, a thermal viable count method was able to quantify microbes coated on seeds despite the presence of the natural microbiota of the seeds. Our results also showed that, in contrast to plating techniques for which clustered cells skew the results, IMC does not require single cells for accurate viable counts. CONCLUSIONS: Thermal viable count methods are novel methods for the rapid quantification of divergent bacterial and fungal species and enhance the speed, sensitivity, and accuracy of routine viable counts of pure cultures and controlled microbiomes such as plant seed coatings.

Comparative assessment of methicillin resistant Staphylococcus aureus diagnostic assays for use in resource-limited settings.

BACKGROUND: The rise of methicillin-resistant Staphylococcus aureus (MRSA) is a global health concern. Paucity of data on MRSA carriage prevalence and diagnostic methods in resource-limited settings hampers efforts to define the problem and plan an appropriate response. Additionally, high variability in cost and logistical characteristics of MRSA screening methods may impede infection control efforts. We compared the performance of locally-available chromogenic agar BD CHROMagar MRSA II and two PCR-based assays (Hain GenoQuick MRSA and Cepheid Xpert SA Complete) for the detection of asymptomatic MRSA carriage in nasal swabs. RESULTS: During 2015, we enrolled 500 patients from five hospital wards at a Ugandan regional referral hospital. We found 30% prevalence of methicillin-sensitive Staphylococcus aureus (MSSA) nasal carriage, and 5.4% MRSA nasal carriage prevalence. Compared to a composite reference standard defined as a positive test result on any one of the three assays, Hain GenoQuick MRSA demonstrated the highest sensitivity (96%) followed by direct plating on CHROMagar at (70%), with the lowest sensitivity observed with Xpert SA Complete (52%). Cepheid Xpert provided the most rapid results (< 1 h) but was the most expensive (US $45-50/test). Substantially more labor was required for the Hain GenoQuick MRSA compared to Xpert SA Complete or CHROMagar tests. CONCLUSION: MRSA nasal carriage prevalence rates were low, and high diagnostic sensitivity was achieved using Hain GenoQuick MRSA. Chromogenic media had significantly lower sensitivity, but may represent a viable local option given its lower cost compared to PCR-based assays.

Microbial study of household hygiene conditions and associated Listeria monocytogenes infection risks for Peruvian women.

OBJECTIVES: To develop an exposure and risk assessment model to estimate listeriosis infection risks for Peruvian women. METHODS: A simulation model was developed utilising Listeria monocytogenes concentrations on kitchen and latrine surfaces in Peruvian homes, hand trace data from Peruvian women and behavioural data from literature. Scenarios involving varying proportions of uncontaminated, or 'clean', surfaces and non-porous surfaces were simulated. Infection risks were estimated for 4, 6 and 8 h of behaviours and interactions with surfaces. RESULTS: Although infection risks were estimated across scenarios for various time points (e.g. 4, 6, 8 h), overall mean estimated infection risks for all scenarios were ≥ 0.31. Infection risks increased as the proportions of clean surfaces decreased. Hand-to-general surface contacts accounted for the most cumulative change in L. monocytogenes concentration on hands. CONCLUSIONS: In addition to gaining insights on how human behaviours affect exposure and infection risk, this model addressed uncertainties regarding the influence of household surface contamination levels. Understanding the influence of surface contamination in preventing pathogen transmission in households could help to develop intervention strategies to reduce L. monocytogenes infection and associated health risks.

OBJECTIFS: Développer un modèle d'exposition et d'évaluation des risques pour estimer les risques d'infection par la listériose chez les femmes péruviennes. MÉTHODES: Un modèle de simulation a été développé en utilisant des concentrations de Listeria monocytogenes sur la surface des cuisines et des latrines dans des foyers péruviens, des données de traces de mains de femmes péruviennes et des données comportementales de la littérature. Des scénarios impliquant différentes proportions de surfaces non contaminées ou «propres¼ et de surfaces non poreuses ont été simulés. Les risques d'infection ont été estimés pour 4, 6 et 8 heures de comportements et d'interactions avec les surfaces. RÉSULTATS: Bien que les risques d'infection aient été estimés pour tous les scénarios à différents moments (par ex. à 4, 6 ou 8 heures), les risques d'infection globaux moyens estimés pour tous les scénarios étaient ≥ 0,31. Les risques d'infection augmentaient à mesure que les proportions de surfaces propres diminuaient. Les contacts entre la main et les surfaces générales contribuent pour le plus de changement cumulatif de la concentration de L. monocytogenes sur les mains. CONCLUSIONS: En plus de comprendre comment les comportements humains affectent l'exposition et le risque d'infection, ce modèle a traité des incertitudes quant à l'influence des niveaux de contamination des surfaces ménagers. Comprendre l'influence de la contamination de surface dans la prévention de la transmission d'agents pathogènes dans les ménages pourrait aider à développer des stratégies d'intervention pour réduire l'infection à L. monocytogenes et les risques associés pour la santé.

Evaluation of the complexity of indoor air in hospital wards based on PM2.5, real-time PCR, adenosine triphosphate bioluminescence assay, microbial culture and mass spectrometry.

BACKGROUND: The aim of this study was to establish a set of assessment methods suitable for evaluating the complex indoor environment of hospital wards and to ascertain the composition of bacteria and microbial ecology of hospital wards. METHODS: Colony-forming units (CFUs), PM2.5 detection, real-time PCR, and adenosine triphosphate (ATP) bioluminescence assay were employed to evaluate the complexity of indoor air in 18 wards of nine departments in a hospital and two student dormitories in a university. Subsequently, the microbial samples were quantified and identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). RESULTS: Although the studied indices were relatively independent, the PM2.5 content was correlated with bacterial CFUs determined by passive sedimentation method, bacterial and fungal counts measured by real-time PCR, and ATP bioluminescence assay. The composition of microorganisms in the air of hospital wards differed from that in the air of student dormitories. The dominant genera in hospital wards were Staphylococcus (39.4%), Micrococcus (21.9%), Corynebacterium (11.7%), Kocuria (4.4%), Bacillus (2.9%), Streptococcus (1.6%), Moraxella (1.6%), and Enterococcus (1.3%), and the microbial ecology differed between Respiration Dept. III and other hospital departments. Additionally, 11.1 and 27.3% of bacteria in hospital wards and student dormitories were not identified, respectively. CONCLUSIONS: Assessment of environmental quality of hospital wards should be based on comprehensive analysis with multiple indicators. There may be imbalances in the microbial diversity in the hospital wards, therefore, monitoring of the environmental quality of hospitals is important in the prevention of nosocomial infections.

Effect of bisphenol-A (BPA) on placental biomarkers for inflammation, neurodevelopment and oxidative stress.

Background Bisphenol-A (BPA) is a widespread pollutant whose effects on pregnant women are poorly understood. Therefore, we investigated the effects of BPA on basal and bacteria-stimulated production of proinflammatory cytokines [interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) and IL-6], anti-inflammatory mediators [soluble glycoprotein 130 (sgp) 130, heme oxidase-1 (HO-1) and IL-10] and biomarkers for neurodevelopment [brain-derived neurotrophic factor (BDNF)], and oxidative stress [8-isoprostane (8-IsoP)] by the placenta. Methods Placental explant cultures were treated with BPA (0-10,000 nM) in the presence or absence of 107 colony-forming unit (CFU)/mL heat-killed Escherichia coli for 24 h. Biomarker concentrations in conditioned medium were quantified by the enzyme-linked immunosorbent assay (ELISA). Results Under basal conditions, IL-1ß and IL-6 production was enhanced by BPA in a dose-dependent manner. Sgp130, a soluble receptor that reduces IL-6 bioactivity, was suppressed by BPA at 1000-10,000 nM. BPA also enhanced BDNF production at 1000 and 10,000 nM, and 8-IsoP expression at 10 and 100 nM. For bacteria-treated cultures, BPA increased IL-6 production at 100 nM and reduced sgp130 at 1000 nM but had no effect on IL-1ß, TNF-α, BDNF, HO-1, 8-IsoP or IL-10 production. Conclusion BPA may increase placental inflammation by promoting IL-1ß and IL-6 but inhibiting sgp130. It may also disrupt oxidative balance and neurodevelopment by increasing 8-IsoP and BDNF production.

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.

Antimicrobial Effect of Picea abies Extracts on E. coli Growth.

This study aims to investigate the effect of essential oils extracted from wood residues of Picea abies on the growth of Escherichia coli. The essential oils were extracted by supercritical carbon dioxide, leading to a yield of 3.4 ± 0.5% (w/w) in 120 min. The antimicrobial effect was tested at 37 °C by isothermal calorimetry. The heat-flow (dq/dt vs. time) was integrated to give a fractional reaction curve (α vs. time). Such curves were fitted by a modified Gompertz function to give the lag-time (λ) and the maximum growth rate (µmax) parameters. The results showed that λ was linearly correlated with E. coli concentration (λ = 1.4 h/log (CFU/mL), R2 = 0.997), whereas µmax was invariant. Moreover, the overall heat was nearly constant to all the dilutions of E. coli. Instead, when the essential oil was added (with concentrations ranging from 1 to 5 mg/L) to a culture of E. coli (104 CFU/mL), the lag-time increased from 14.1 to 33.7 h, and the overall heat decreased from 2120 to 2.37 J. The results obtained by the plate count technique were linear with the lag-time (λ), where (λ = -7.3 × log (CFU/mL) + 38.3, R2 = 0.9878). This suggested a lower capacity of E. coli to metabolize the substrate in the presence of the essential oils. The results obtained in this study promote the use of essential oils from wood residues and their use as antimicrobial products.

Statistical Package for Growth Rates Made Easy.

Growth rates are an important tool in microbiology because they provide high throughput fitness measurements. The release of GrowthRates, a program that uses the output of plate reader files to automatically calculate growth rates, has facilitated experimental procedures in many areas. However, many sources of variation within replicate growth rate data exist and can decrease data reliability. We have developed a new statistical package, CompareGrowthRates (CGR), to enhance the program GrowthRates and accurately measure variation in growth rate data sets. We define a metric, Variability-score (V-score), that can help determine if variation within a data set might result in false interpretations. CGR also uses the bootstrap method to determine the fraction of bootstrap replicates in which a strain will grow the fastest. We illustrate the usage of CGR with growth rate data sets similar to those in Mira, Meza, et al. (Adaptive landscapes of resistance genes change as antibiotic concentrations change. Mol Biol Evol. 32(10): 2707-2715). These statistical methods are compatible with the analytic methods described in Growth Rates Made Easy and can be used with any set of growth rate output from GrowthRates.

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.

PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media.

BACKGROUND: Microbial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways. RESULTS: The "PHENOS" pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive. CONCLUSIONS: "PHENOS" is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos .