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bioRxiv2019

Cryptic role of tetrathionate in the sulfur cycle: A study from Arabian Sea oxygen minimum zone sediments

To explore the potential role of tetrathionate in the sulfur cycle of marine sediments, the population ecology of tetrathionate-forming, oxidizing, and respiring microorganisms was revealed at 15-30 cm resolution along two, [~]3-m-long, cores collected from 530- and 580-mbsl water-depths of Arabian Sea, off Indias west coast, within the oxygen minimum zone (OMZ). Metagenome analysis along the two sediment-cores revealed widespread occurrence of the structural genes that govern these metabolisms; high diversity and relative-abundance was also detected for the bacteria known to render these processes. Slurry-incubation of the sediment-samples, pure-culture isolation, and metatranscriptome analysis, corroborated the in situ functionality of all the three metabolic-types. Geochemical analyses revealed thiosulfate (0-11.1 M), pyrite (0.05-1.09 wt %), iron (9232-17234 ppm) and manganese (71-172 ppm) along the two sediment-cores. Pyrites (via abiotic reaction with MnO2) and thiosulfate (via oxidation by chemolithotrophic bacteria prevalent in situ) are apparently the main sources of tetrathionate in this ecosystem. Tetrathionate, in turn, can be either converted to sulfate (via oxidation by the chemolithotrophs present) or reduced back to thiosulfate (via respiration by native bacteria); 0-2.01 mM sulfide present in the sediment-cores may also reduce tetrathionate abiotically to thiosulfate and elemental sulfur. Notably tetrathionate was not detected in situ - high microbiological and geochemical reactivity of this polythionate is apparently instrumental in the cryptic nature of its potential role as a central sulfur cycle intermediate. Biogeochemical roles of this polythionate, albeit revealed here in the context of OMZ sediments, may well extend to the sulfur cycles of other geomicrobiologically-distinct marine sediment horizons.

microbiology↗

bioRxiv2019

Evaluation of waste paper for cultivation of oyster mushroom (Pleurotus ostreatus) with some added supplementary materials

Mushroom cultivation is an economically feasible bio-technological process for conversion of various lignocellulosic wastes. This study was conducted at Aksum University with the aim of evaluating the suitability of waste paper supplemented with corn stalk and wheat bran for Oyster mushroom cultivation. Spawn were prepared in Microbiology laboratory and inoculated into the prepared substrates. Waste paper supplemented with corn stalk and wheat bran with 0%, 25% and 50% were tested for their productivity and biological efficiency (BE) for cultivation of P.ostreatus mushroom. Data were analyzed using SPSS version 20. Higher (26.20{+/-} 19.36) mean weight, pileus diameter (7.90 {+/-}2.66cm), total yield (646.4 {+/-}273.1gm) and BE (64.64{+/-} 273 % were obtained from waste paper (50%) +cornstalk (25%) +wheat bran (25%). However, Lower (17.92{+/-}81.95%) BE were obtained from waste paper (100%). Moreover, the highest (3.88 {+/-}0.32 cm) mean stalk length were obtained from waste paper (50%) + cornstalk (50). This study revealed that waste paper supplemented with corn stalk and wheat bran results in high BE and total yield. Thus, appears to be a promising alternative for the cultivation of oyster mushroom. Yet, waste paper without supplement poorly supports the growth of P.ostreatus mushroom.

microbiology↗

bioRxiv2019

Prevalence of Chlamydia trachomatis and Candida albicans; hospital based study

BackgroundChlamydia and candidiasis have only mild or no symptoms at all. When symptoms develop, they are often mistaken for something else like urinary tract infections or yeast infections. These infections affect both men and woman of all backgrounds and economic levels.\n\nObjectiveThe prevalence of Chlamydia trachomatis (C.trachomatis) and Candida albicans (C.albicans) infections among attendees of Kilifi Medical Centre in Kilifi county, Kenya was studied.\n\nMethodologyUrethral and vaginal swabs samples were aseptically collected from 305 subjects, examined for Candida albicans and Chlamydia trachomatis using standard microbiological methods. The swabs were analyzed using direct wet smears, Gram-stained smear and culture techniques.\n\nResultsOf the 305 participants, 181 (59.34%) females and 124 (40.66%) males were tested with the overall prevalence of 53.44 % for both Chlamydia trachomatis and Candida albicans with females having a higher infection rate (35.14 %) for chlamydia and candidasis than men (17.71). Amongst the different age groups investigated, candida and chlamydia distribution was highest in participants aged 28-32 years (21.97 %).The infection rate of C.trachomatis (14.43 %) among the male participants was higher than the infection rate revealed among the female participants of 1.97 % while the infection rate of C.albicans was higher among the female participants (33.77 %) compared to the 3.28 % recorded in male participants with no co-infections revealed.\n\nConclusionThe results of this study demonstrated a significant difference between male and female chlamydia and candida infections with women being severely affected than men. The study recommended routine screening for sexually transmitted infections (STIs) which is essential in preventing infections transmissions, assessment of the role of socio-demographic and behavioral risks on Chlamydia trachomatis and Candida albicans, proper treatment of all candida and chlamydia by use of correct/effective medicines, contact tracing and treatment of sexual partners and health education.

microbiology↗

bioRxiv2019

Multi-drug resistance bacteria predict mortality in blood stream infection in a tertiary setting in Tanzania

BackgroundBlood-stream infections (BSIs) are serious and life-threatening infections associated with high mortality and morbidity. In resource limited settings, there is paucity of data on predictors of outcome in patients with BSI. This study aimed at examining the predictors of mortality in patients with BSI as well as bacteria causing BSI.\n\nMethods and MaterialsThis was cross-sectional study conducted in Muhimbili National Hospital between April and May 2018. Blood culture results from all inpatients at clinical microbiology laboratory were recorded and clinical information were retrieved retrospectively from the files. Bacteria from positive blood culture were identified and antimicrobial susceptibility was performed.\n\nResultsThe overall prevalence of BSI was 11.4% (46/402), with case fatality rate of 37%. There was significant high rate of BSI in patient who had died compared to those survived p= 0.008. Gram-negative bacteria (74%) were the common cause of BSI, with predominance of Enterobacteriaceae (22), followed by Pseudomonas aeruginosa (11). Majority (70.5%) of the bacteria isolated from patients with BSI were multi-drug resistant. Forty six percent of Pseudomonas aeruginosa were resistance to meropenem. Sixty eight percent 68.2% (15/22) of Enterobacteriaceae were ESBL producers. Carbapenemases production were detected in 27% (3/11) of Pseudomonas aeruginosa and in one Proteus mirabillis. Forty percent (40%) of Staphylococcus aureus were methicillin resistant Staphylococcus aureus (MRSA). Positive blood culture (cOR 7.4, 95%CI 1.24 - 43.83, p 0.03) and admission in ICU cOR 4 (95%CI 1.7 - 9.41, p 0.001) were independent factors for mortality in suspected BSI. Isolation of Multi-drug resistant bacteria was independent predictor for mortality in confirmed BSI (cOR 7.4, 95%CI 1.24 - 43.83, p 0.03).\n\nConclusionThe prevalence of BSI was 11.4%, with majority of bacteria in BSI were MDR. Positive blood culture and MDR were predictors for mortality.

microbiology↗

bioRxiv2019

Targeted metagenomic sequencing enhances the identification of pathogens associated with acute infection.

The routine identification of pathogens during infection remains challenging because it relies on multiple modalities such as culture and nucleic acid amplification and tests that tend to be specific for very few of an enormous number of possible infectious agents. Metagenomics promises single-test identification, but shotgun sequencing remains unwieldy and expensive or in many cases insufficiently sensitive to detect the amount of pathogen material in a clinical sample. Here we present the validation and application of Castanet, a method for metagenomic sequencing with enrichment that exploits clinical knowledge to construct a broad panel of relevant organisms for detection at low cost with sensitivity comparable to PCR. Castanet targets both DNA and RNA, works with small sample volumes, and can be implemented in a high-throughput diagnostic setting. We used Castanet to analyse plasma samples from 573 patients from the GAinS sepsis cohort and CSF samples from 243 patients from the ChiMES meningitis cohort that had been evaluated using standard clinical microbiology methods, identifying relevant pathogens in many cases where no pathogen had previously been detected. Castanet is intended for use in defining the distribution of pathogens in samples, diseases and populations, for large-scale clinical studies and for verifying the performance of routine testing regimens. By providing sequence as output, Castanet combines pathogen identification directly with subtyping and phylo-epidemiology.

microbiology↗

bioRxiv2019

Microbial community dynamics of surface water in British Columbia, Canada

Traditional methods for monitoring the microbiological quality of water focus on the detection of fecal indicator bacteria such as Escherichia coli, often tested as a weekly grab sample. To understand the stability of E.coli concentrations over time, we evaluated three approaches to measuring E. coli levels in water: microbial culture using Colilert, quantitative PCR for uidA and next-generation sequencing of the 16S rRNA gene. Two watersheds, one impacted by agricultural and the other by urban activities, were repeatedly sampled over a simultaneous ten-hour period during each of the four seasons. Based on 16S rRNA gene deep sequencing, each watershed showed different microbial community profiles. The bacterial microbiomes varied with season, but less so within each 10-hour sampling period. Enterobacteriaceae comprised only a small fraction (<1%) of the total community. The qPCR assay detected significantly higher quantities of E. coli compared to the Colilert assay and there was also variability in the Colilert measurements compared to Health Canadas recommendations for recreational water quality. From the 16S data, other bacteria such as Prevotella and Bacteroides showed promise as alternative indicators of fecal contamination. A better understanding of temporal changes in watershed microbiomes will be important in assessing the utility of current biomarkers of fecal contamination, determining the best timing for sample collection, as well as searching for additional microbial indicators of the health of a watershed.

microbiology↗

bioRxiv2019

The concentration of heterotrophic growth substrates in culture medium is a crucial determinant of the culturability of subsurface soil microbes

The vast majority of microbes inhabiting oligotrophic shallow subsurface soil environments have not been isolated or studied under controlled laboratory conditions. In part, the challenges associated with isolating shallow subsurface microbes may persist because microbes in deeper soils are adapted to low nutrient availability or quality. Here we use high-throughput dilution-to-extinction culturing to isolate shallow subsurface microbes from a conifer forest in Arizona, USA. We hypothesized that the concentration of heterotrophic substrates in microbiological growth medium would affect which microbial taxa were culturable from these soils. To test this, we diluted extracted cells into one of two custom-designed defined growth media that differed only by a 100-fold difference in the concentration of amino acids and organic carbon. Across both media, we isolated a total of 133 pure cultures, all of which were classified as Actinobacteria or Alphaproteobacteria. The substrate availability dictated which actinobacterial phylotypes were culturable but had no significant effect on the culturability of Alphaproteobacteria. We isolated cultures that were representative of the most abundant phylotype in the soil microbial community (Bradyrhizobium spp.) and representatives of five of the top 10 most abundant Actinobacteria phylotypes, including Nocardioides spp., Mycobacterium spp., and several other phylogenetically-divergent lineages. Flow cytometry of nucleic acid-stained cells showed that cultures isolated on low-substrate medium had significantly lower nucleic-acid fluorescence than those isolated on high-substrate medium. These results show that dilution-to-extinction is an effective method to isolate abundant soil microbes and the concentration of substrates in culture medium influences the culturability of specific microbial lineages. ImportanceIsolating environmental microbes and studying their physiology under controlled conditions is an essential aspect of understanding their ecology. Subsurface ecosystems are typically nutrient-poor environments that harbor diverse microbial communities--the majority of which are thus far uncultured. In this study, we use modified high-throughput cultivation methods to isolate subsurface soil microbes. We show that a component of whether a microbe is culturable from subsurface soils is the concentration of growth substrates in the culture medium. Our results offer new insight into technical approaches and growth medium design that can be used to access the uncultured diversity of soil microbes.

microbiology↗

bioRxiv2019

PCR-based detection of Helicobacter spp. in mice from different animal houses in Rio de Janeiro, Brazil

Pathogenic microbial detection and control in breeding and experimental laboratory animal facilities is essential to guarantee animal welfare, data validity and reproducibility. Helicobacter spp. is known to severely affect mice health, mainly in immunocompromised strains, what may affect experimental outcomes. This study aimed to screen for Helicobacter spp. in mice from four different animal houses in Rio de Janeiro, Brazil using a PCR for 16S ribosomal RNA. A pair of primers was designed to specifically identify Helicobacter species that commonly infect laboratory mice. Following PCR reaction, the expected 375 base pairs (bp) amplification product was purified, sequenced and showed a 95% similarity when compared to deposited sequences of Helicobacter hepaticus and Helicobacter bilis. Then, the presence of Helicobacter spp. in both feces and intestines samples was analyzed. Helicobacter spp DNA was detected in 59.6% of the fecal and 70.17% of the intestine samples. Although Helicobacter spp screening is recommended by institutional animal health monitoring programs worldwide it is still not mandatory by Brazilian animal welfare regulation. Our study, the first to monitor Helicobacter species in laboratory mice in Brazil, demonstrates the possibility of using a low cost, rapid molecular diagnostic test to screen Helicobacter and highlights the importance of regular microbiological verification of mice used for research in Brazilian animal houses.

microbiology↗

bioRxiv2019

Exploring the microbial diversity in oil-contaminated mangrove sediments using 16S rRNA metagenomics

With an area of 390 hectares, the mangrove forests of Nayband Bay are the widest mangrove communities above 27 degrees latitude in the Persian Gulf. They are the last dense, extensive structures of these ecosystems found in the north-west Indian Ocean. Growing industrial activities near the Nayband Bay and the consequent marine pollution has resulted in damage and threatened different marine habitats in recent years. To date, a comprehensive description of the microbial life in the mangrove ecosystem that has been exposed to oil contamination is lacking. This information could significantly contribute to a better overview of the function and resilience of the ecosystem. This work represents a first effort to better understand the Nayband Bay mangrove microbiology by applying 16S rRNA metagenomics. A total of 65,408 readings from the V3-V4 16S rRNA gene regions were obtained from 24 sediment samples, each measuring 440 bp. Most sequences belonged to members of the Proteobacteria phylum (mainly {gamma}-Proteobacteria); however, members of the Bacteroidetes phylum (mainly Flavobacteriia) were also well represented in the samples. We discovered that the community of this ecosystem strongly exhibit typical structures of oil-contaminated marine environments. This is likely due to the growing industrial activity in the area and its consequent marine polluting effects. The use of practicable and applicable bioremediation protocols for habitat restoration in this valuable area is needed.

microbiology↗

bioRxiv2019

External quality assessment of bacterial identification and antimicrobial susceptibility testing in African national public health laboratories, 2011-2016

BackgroundIn 2002, the World Health Organization (WHO) launched a regional microbiology external quality assessment (EQA) programme for national public health laboratories in the African region, initially targeting priority epidemic-prone bacterial diseases, and later including other common bacterial pathogens.\n\nObjectivesThe aim of this study was to analyse the efficacy of an EQA programme as a laboratory quality system evaluation tool.\n\nMethodsWe analysed the proficiency of laboratories performance of bacterial identification and AST for the period 2011-2016. Overall, 81 laboratories from 45 countries participated.\n\nThe National Institute for Communicable Diseases of South Africa provided technical coordination following an agreement with WHO, and supplied EQA samples of selected bacterial organisms for microscopy (Gram stain), identification, and antimicrobial susceptibility testing (AST). National public health laboratories, as well as laboratories involved in the Invasive Bacterial Diseases Surveillance Network, were enrolled by the WHO Regional Office for Africa to participate in the EQA programme.\n\nResultsWe analysed participants results of 41 surveys, which included the following organisms sent as challenges: Streptococcus pneumonia, Haemophilus influenza, Neisseria meningitidis, Salmonella Typhi, Salmonella Enteritidis, Shigella flexneri, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus anginosus, Enterococcus faecium, Serratia marcescens, Acinetobacter baumannii, and Enterobacter cloacae. Overall, 76% of participants obtained acceptable scores for identification, but a substantial proportion of AST scores were not in the acceptable range.\n\nConclusionIn the African Region, implementation of diagnostic stewardship in clinical bacteriology is generally suboptimal. This report illustrates that AST is poorly done compared to microscopy and identification. It is critically important to make the case for implementation of quality assurance in AST, as it is the cornerstone for antimicrobial resistance surveillance reporting and implementation of the Global Antimicrobial Resistance Surveillance System.

microbiology↗

bioRxiv2019

Fast and robust detection of colistin resistance in Escherichia coli using the MALDI Biotyper Sirius mass spectrometry system

Polymyxin antibiotics are a last-line treatment for multidrug-resistant Gram-negative bacteria. However, the emergence of colistin resistance, including the spread of mobile mcr genes, necessitates the development of improved diagnostics for the detection of colistin-resistant organisms in hospital settings. The recently developed MALDIxin test enables detection of colistin resistance by MALDI-TOF mass spectrometry in less than 15 minutes but is not optimized for the mass spectrometers commonly found in clinical microbiology laboratories. In this study, we adapted the MALDIxin test for the MALDI Biotyper Sirius MALDI-TOF mass spectrometry system (Bruker Daltonics). We optimized the sample preparation protocol using a set of 6 MCR-expressing Escherichia coli clones and validated the assay with a collection of 40 E. coli clinical isolates, including 19 MCR producers, 12 chromosomally-resistant isolates and 9 polymyxin-susceptible isolates. We calculated Polymyxin resistance ratio (PRR) values from the acquired spectra; a PRR value of zero, indicating polymyxin susceptibility, was obtained for all colistin-susceptible E. coli isolates, whereas positive PRR values, indicating resistance to polymyxins, were obtained for all resistant strains independent of the genetic basis of resistance. Thus, we report a preliminary feasibility study showing that an optimized version of the MALDIxin test, adapted for the routine MALDI Biotyper Sirius, provides an unbiased, fast, reliable, cost-effective and high-throughput way of detecting colistin resistance in clinical E. coli isolates.

microbiology↗

bioRxiv2019

High-throughput isolation and sorting of gut microbes reduce biases of traditional cultivation strategies

Traditional cultivation approaches in microbiology are labor-intensive, low-throughput, and often yield biased sampling of taxa due to ecological and evolutionary factors. New strategies are needed to enable ample representation of rare taxa and slow-growers that are outcompeted by fast-growing organisms. We developed a microfluidic platform that anaerobically isolates and cultivates microbial cells in millions of picoliter droplets and automatically sorts droplets based on colony density. We applied our strategy to mouse and human gut microbiomes and used 16S ribosomal RNA gene amplicons to characterize taxonomic composition of cells grown using different media. We found up to 4-fold increase in richness and larger representation of rare taxa among cells grown in droplets compared to conventional culture plates. Automated sorting of droplets for slow-growing colonies further enhanced the relative abundance of rare populations. Our method improves the cultivation and analysis of diverse microbiomes to gain deeper insights into microbial functioning and lifestyles.

microbiology↗

bioRxiv2019

Within-Host Diversity and Vertical Transmission of Group B Streptococcus Among Mother-infant Dyads in The Gambia

IntroductionUnderstanding mother-to-infant transmission of Group B Streptococcus (GBS) is vital to the prevention and control of GBS disease. We investigated the transmission and phylogenetic relationships of mothers colonised by GBS and their infants in a peri-urban setting in The Gambia.\n\nMethodsWe collected nasopharyngeal swabs from 35 mother-infant dyads at weekly intervals from birth until six weeks post-partum. GBS was isolated by conventional microbiology techniques. Whole-genome sequencing was performed on GBS isolates from one mother-infant dyad (dyad 17).\n\nResultsWe recovered 85 GBS isolates from the 245 nasopharyngeal swabs. GBS was isolated from 16.33% and 18.37% of sampled mothers and infants, respectively. In 87% of cultured swabs, the culture status of an infant agreed with that of the mother (Kappa p-value <0.001). In dyad 17, phylogenetic analysis revealed within-host strain diversity in the mother and clone to her infant.\n\nConclusionGBS colonisation in mothers presents a significant risk of colonisation in their infants. We confirm vertical transmission from mother to child in dyad 17, accompanied by within-host diversity.

microbiology↗

bioRxiv2019

Three-dimensional label-free observation of individual bacteria upon antibiotic treatment using optical diffraction tomography

Measuring alterations in bacteria upon antibiotic application is important for basic studies in microbiology, drug discovery, and clinical diagnosis, and disease treatment. However, imaging and 3D time-lapse response analysis of individual bacteria upon antibiotic application remain largely unexplored mainly due to limitations in imaging techniques. Here, we present a method to systematically investigate the alterations in individual bacteria in 3D and quantitatively analyze the effects of antibiotics. Using optical diffraction tomography, in-situ responses of Escherichia coli and Bacillus subtilis to various concentrations of ampicillin were investigated in a label-free and quantitative manner. The presented method reconstructs the dynamic changes in the 3D refractive-index distributions of living bacteria in response to antibiotics at sub-micrometer spatial resolution.

microbiology↗

bioRxiv2019

An analysis and optimization of growth condition requirements of the fast-growing bacterium Vibrio natriegens

The fast-growing Gram-negative bacterium Vibrio natriegens is an attractive host for a range of applications in molecular biology and biotechnology. Moreover, the remarkable speed of growth of Vibrio natriegens poses fundamental questions on bacterial physiology and metabolism, energy production, DNA replication and protein synthesis, besides others. In order to address such questions, a solid understanding of the physiological and physical/chemical basis of growth requirements is essential. Here we report a systematic analysis of i) various growth media composition, ii) incubation temperature, iii) pH dependence, and iv) salt concentration requirements for optimal growth of V. natriegens strain DSMZ 759. As a result of the studies, the following optimal conditions were Established: LB medium with 2.5 % NaCl, pH 7.0 - 8.5 and incubation at 37{degrees}C under aerobic conditions. Incubation temperatures above 37 {degrees}C slows growth significantly. Incubation temperatures below 37 {degrees}C slows growth, but at a lower rate. Incubation at or below 28 {degrees}C should be avoided. Under such optimized, standard laboratory conditions, a doubling time of td = 13.6 minutes was observed for V. natriegens measured in mid-log growth phase. The optimized conditions presented here for the growth of V. natriegens can be easily applied in any standardly equipped laboratory. For comparison, identical growth conditions for Escherichia coli were analyzed and are presented as well.\n\nIMPORTANCEGoal of this study was to understand the physiological growths requirements of V. natriegens in routine microbiology and molecular biology laboratory settings. The result is a standardized protocol for the optimized growth of the naturally isolated (wild type) V. natriegens strain DSMZ 759. This protocol can be employed for routine application of V. natriegens for any kind of biochemical, molecular biology and genomic studies and utilization under normal laboratory conditions used by many routinely equipped laboratories.

microbiology↗

bioRxiv2019

Relevance of screening for Chagas and viral hepatitis in Bolivian migrants

Objectivesgiven the scarcity of data regarding prevalence of various infectious diseases in Latin-American countries, our study aims to assess the burden of T.cruzi, S.stercoralis, HIV and viral hepatitis in Latin-American migrants, with a focus on Bolivian migrants.\n\nMethodswe performed a retrospective observational study of 565 screening evaluations on adults ([≥] 18 years) carried out at our referral International Healthcare service in Barcelona. We reviewed structured clinical records and microbiological results of patients attended between February 2012 and April 2015.\n\nResultsthe median 35 years old and 74% were women. Bolivian origin accounted for 87% of the screened population. We found a 48% prevalence of T.cruzi, 16% of S.stercoralis, 0.2% of HIV, 92% of HAV, 0.2% HBV and 0.2% HCV.\n\nConclusionsthese results support the relevance of the screening of T. cruzi and S. stercoralis in Bolivian migrants, but challenge the pertinence of systematic screening of HBV in this population.\n\nAuthor summaryIn response to the challenge of detecting diseases not previously present in host countries, screening programs have been implemented for migrants based on the probability of having certain diseases depending on their country of origin and / or migratory route. This increased risk is very clearly established in some cases such as Trypanosoma cruzi infection (the cause of Chagas disease) in people from Latin America; especially from Bolivia. In recent years screening recommendations for Strongyloides stercoralis in this population was proven necessary. Current recommendations regarding systematic screening for hepatitis B establish the relevance of screening based on the probability of the disease in the 2% population of origin. Since there are no reliable and up to date data regarding prevalence of hepatitis B virus in Bolivia, we aimed to analyze data available for migrants from Bolivia in Spain.\n\nOur results support the importance of screening for T. cruzi and S.stercoralis in patients from Bolivia. However, our data show a much lower prevalence of this hepatitis B virus (0.2%) than the 2% threshold that would justify systematic screening, so we question the relevance of screening for hepatitis B virus in this population in the absence of other risk factors.

microbiology↗

bioRxiv2019

Prospective evaluation of beta-lactamase detection in penicillin susceptible Staphylococcus aureus by interpretation of the penicillin disc edge

Penicillin susceptible Staphylococcus aureus (PSSA) may occasionally be encountered as a cause of complicated S. aureus infection, such as endocarditis or bloodstream infections. Clinicians may choose to treat these patients with penicillin over a semi-synthetic penicillin derivative, such as flucloxacillin or oxacillin, due to a favourable Pk/Pd profile. In this study, we prospectively evaluated the penicillin disc (1-IU) method for detection of blaZ, with interpretation of the penicillin edge according to EUCAST recommendations. 472 PSSA isolates were collected between September 2014 to December 2015 from three clinical microbiology laboratories in Queensland, Australia. Initial antimicrobial susceptibility testing was performed by the Vitek 2 system. Real-time PCR for blaZ was performed following phenotypic testing with the 1-IU penicillin disc and the PCR used as the gold standard for detection of penicillinase. The prevalence of blaZ amongst the isolates was 7%. The sensitivity, specificity, positive predictive value and negative predictive value of the penicillin disc method was 97%, 95%, 61% and 100% when compared to blaZ PCR. In summary, the penicillin disc zone size and edge interpretation is a reliable method for detection of blaZ in S. aureus isolates that otherwise test susceptible to penicillin by Vitek 2 AST.

microbiology↗

bioRxiv2019

Flaming as part of aseptic technique increases CO2(g) and decreases pH in freshwater culture media

Aseptic technique has historically served as a fundamental practice in microbiology, helping maintain culture purity and integrity. This technique has been widely encouraged and employed for use with cultures of heterotrophic bacteria as well as freshwater and marine algae. Yet, recent observations have suggested these approaches may bring their own influences. We observed variations in growth amongst replicate experimental cyanobacterial cultures upon flaming of the culture tube opening during sample transfer and collection. Investigation revealed the pH of culture media had decreased from the initial pH established during media preparation. Flaming of sterile culture media alone confirmed a significant decrease, by as much as 1.7 pH units, and correlated with increased flaming events over time. We hypothesized that the causative factor was the introduction of carbon dioxide into the media. To test this hypothesis, qualitative and quantitative analyses were used to identify the primary driver of pH decline. We further assessed the direct effects of flaming and subsequent pH changes on Microcystis aeruginosa cultures, showing flame-driven pH changes and/or the introduction of carbon dioxide influenced experimental results. Our observations provide a cautionary tale of how classic and well-accepted approaches may not always provide the results promised, suggesting new approaches may be necessary in research areas assessing pH or carbon related-effects on microbial communities.

microbiology↗