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bioRxiv2019

Non-Invasive Detection of Viral Antibodies Using Oral Flocked Swabs

Salivary antibodies are useful in surveillance and vaccination studies. However, low antibody levels and degradation by endonucleases are problematic. Oral flocked swabs are a potential non-invasive alternative to blood for detecting viral antibodies. Serum and saliva collected from 50 healthy volunteers were stored at -80{degrees}C; dried swabs at room temperature. Seroprevalence for Cytomegalovirus (CMV), Varicella-Zoster virus (VZV), Epstein-Barr virus (EBV), Measles and Mumps IgG antibodies were determined using commercial ELISAs and processed on an automated platform. For each antibody, swabs correlated well with saliva. For CMV IgG, VZV IgG, and EBV EBNA-1 IgG and VCA IgG, the swab sensitivities compared to serum were 95.8%, 96%, 92.1% and 95.5% respectively. For Measles IgG, swab sensitivity was 84.5%. Mumps IgG displayed poor sensitivity for oral swabs (60.5%) and saliva (68.2%). Specificities for IgG antibodies were 100% for CMV, EBV and Mumps. Specificities for VZV and Measles could not be determined due to seropositive volunteers. As oral flocked swabs correlate well with serum, are easy to self-collect and stable at room temperature further research is warranted. HighlightsO_LIOral flocked swabs are an easy, self-collection method for measuring viral antibodies. C_LIO_LIViral IgG is stable on dried oral flocked swabs for at least two years. C_LIO_LIOral swabs are highly sensitive for CMV, VZV, and EBV IgG. C_LIO_LIOral swabs are potentially useful for surveillance and clinical microbiology. C_LI

microbiology↗

bioRxiv2019

Aerobic bacteria as Escherichia coli can survive in ESwabTM medium after a 3 month-freezing at -80°C but not after multiple thawing

Pretesting of procedures for specimen conservation should be part of preliminary studies for trials especially when quantification of bacteria must be performed in a second time. Quantitative epidemiological studies of multidrug resistant organisms sampled from rectal swabs could be then particularly favored. The aim of this preliminary study, was to evaluate the performance of the flocked swab with liquefied Amies transport medium ESwab, for the survival evaluation of aerobic bacteria, from rectal swab sampling according to the number of freezing at -80{degrees}C and thawing cycles and the time of freezing. We first observed that quantification was not reliable after F/T cycles whereas a unique freezing could be performed especially when studying E. coli isolates. The second experiment allowed us to observe that this stability could be obtained until 3 month-freezing. Our study represents a preliminary study, confirming the utility of ESwab in microbiological diagnostics and research studies, not only for molecular bacterial tests, but also, for the maintenance of bacterial viability in clinical specimens.

microbiology↗

bioRxiv2019

Electrically induced bacterial membrane potential dynamics correspond to cellular proliferation capacity

Membrane-potential dynamics mediate bacterial electrical signaling at both intra- and inter- cellular levels. Membrane potential is also central to cellular proliferation. It is unclear whether the cellular response to external electrical stimuli is influenced by the cell proliferative capacity. A new strategy enabling electrical stimulation of bacteria with simultaneous monitoring of single-cell membrane potential dynamics would allow bridging this knowledge gap and further extend electrophysiological studies into the field of microbiology. Here we report that an identical electrical stimulus can cause opposite polarization dynamics depending on cellular proliferation capacity. This was demonstrated using two model organisms, namely B. subtilis and E. coli, and by developing an apparatus enabling exogenous electrical stimulation and single-cell time-lapse microscopy. Using this bespoke apparatus, we show that a 2.5 sec electrical stimulation causes hyperpolarization in unperturbed cells. Measurements of intracellular K+ and the deletion of the K+ channel suggested that the hyperpolarization response is caused by the K+ efflux through the channel. When cells are pre-exposed to UV-violet light, the same electrical stimulation depolarizes cells instead of causing hyperpolarization. A mathematical model extended from the FitzHugh-Nagumo neuron model suggested that the opposite response dynamics are due to the shift in resting membrane potential. As predicted by the model, electrical stimulation only induced depolarization when cells are treated with antibiotics, protonophore or alcohol. Therefore, electrically induced membrane potential dynamics offer a novel and reliable approach for rapid detection of proliferative bacteria and determination of their sensitivity to antimicrobial agents at the single-cell level.

microbiology↗

bioRxiv2019

Comparative study of GenePOC GBS LB Assay and GeneXpert GBS LB Assay for the detection of Group B Streptococcus in prenatal screening samples.

Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC, therefore recommends GBS screening for all pregnant women at 35-37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, GenePOC GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsins clinical microbiology laboratory for GBS testing. We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with higher sensitivity observed for the GenePOC GBS LB assay, and higher specificity for the GeneXpert GBS LB assay.

microbiology↗

bioRxiv2019

Mycobacterium tuberculosis detection from oral swabs with Xpert MTB/RIF ULTRA: A pilot study

ObjectiveDiagnostic testing for tuberculosis depends on microbiological detection of Mycobacterium tuberculosis (Mtb) in sputum. For patients unable to expectorate sputum, such as children and individuals living with HIV, this poses barriers to rapid diagnosis and treatment initiation. Therefore, this study aimed to use oral swabs have as alternative sample type for Mtb detection via molecular testing. ResultsIn a pilot study, we aimed to evaluate sensitivity of Mtb detection via oral swabs using Xpert MTB/RIF ULTRA. We enrolled 63 TB cases and controls from Lima, Peru, and detected Mtb from oral swabs with a sensitivity of 45% (95% confidence interval (CI): 29%-63%) and specificity of 100% (95% CI: 84%-100%) using liquid culture of sputum as reference test. Our current protocol will need optimization, but these results support future exploration of the use of oral swabs for Mtb detection.

microbiology↗

bioRxiv2019

Cross-transmission is not the source of new Mycobacteirum abscessus infections in a multi-centre cohort of cystic fibrosis patients

BackgroundMycobacterium abscessus is an extensively drug resistant pathogen that causes pulmonary disease particularly in cystic fibrosis (CF) patients. Identifying direct patient-to-patient transmission of M. abscessus is critically important in directing infection control policy for the management of risk in CF patients. A variety of clinical labs have used molecular epidemiology to investigate transmission. However there is still conflicting evidence as to how M. abscessus is acquired and whether cross-transmission occurs. Recently labs have applied whole-genome sequencing (WGS) to investigate this further and in this study we investigate whether WGS can reliably identify cross-transmission in M. abscessus.\n\nMethodsWe retrospectively sequenced the whole genomes of 145 M. abscessus isolates from 62 patients seen at four hospitals in two countries over 16 years.\n\nResultsWe have shown that a comparison of a fixed number of core single nucleotide variants (SNVs) alone cannot be used to infer cross-transmission in M. abscessus but does provide enough information to replace multiple existing molecular assays. We detected one episode of possible direct patient-to-patient transmission in a sibling pair. We found that patients acquired unique M. abscessus strains even after spending considerable time on the same wards with other M. abscessus positive patients.\n\nConclusionsThis novel analysis has demonstrated that the majority of patients in this study have not acquired M. abscessus through direct patient-patient transmission or a common reservoir. Tracking transmission using WGS will only realise its full potential with proper environmental screening as well as patient sampling.\n\nKey pointsO_LIWhole genome sequencing should replace current molecular typing used routinely in clinical microbiology laboratories.\nC_LIO_LIPatient-to-patient spread of M. abscessus is not common.\nC_LIO_LIEnvironmental screening may provide a better understanding acquisition of M. abscessus infections.\nC_LI

microbiology↗

bioRxiv2019

Cultivating in situ triggers growth of previously uncultivated microorganisms via a growth initiation factor in nature

Most microorganisms resist cultivation under standard laboratory conditions. On the other hand, to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. In the present study, we applied in situ cultivation to isolate diverse previously uncultivated marine sponge-associated microbes and comparatively analyzed this methods efficiencies with those of the conventional method. Then, we attempted to clarify the key and unknown mechanism of in situ cultivation by focusing on growth triggering via growth initiation factor. We hypothesized that majority of environmental microorganisms are in nongrowing state and requiring \"growth initiation factor\" for the recovery and that can be provided from environments. Consequently, significantly more novel and diverse microbial types were isolated via in situ cultivation than by standard direct plating (SDP). Next, the effect of the sponge extract on starvation recovery was compared between strains derived from in situ and SDP cultivation. Adding small amounts of the sponge extracts to the medium elevated the colony-formation efficiencies of the in situ strains at the starvation recovery step, while it showed no positive effect on that of SDP strains. Conversely, specific growth rates or carrying capacities of all tested strains were not positively affected. These results indicate that, 1) the sponge extract contains chemical compounds that facilitate starvation recovery, these substances selectively worked on the in situ strains, and 2) growth initiation factor in the sponge extract did not continuously promote growth activity but worked as triggers for regrowth (resuscitation from dormancy).\n\nImportanceMost microbial species resist cultivation under laboratory condition. This is critical impediment for both academic and applied microbiology, and thus clarification of the mechanism of microbial uncultivability is highly demanded. Several evidences have been reported that to cultivate microbes in a membrane-bound device incubated in nature (in situ cultivation) is an effective approach. However, the mechanism behind this approach has not been clarified. The present study shows the evidence that 1) initiating growth is a key for cultivating previously uncultivated microbes rather than simple growth promotion, and 2) growth initiation factor (signaling-like compounds) in natural environments stimulate microbial resuscitation from a nongrowing state. Since no study has focused on growth initiation for cultivation of previously uncultivated microorganisms, the discovery shown in the present study provides a new insight into microorganisms previously considered uncultivable and a microbial growth controlling system in nature.

microbiology↗

bioRxiv2019

Domestication of Campylobacter jejuni NCTC 11168

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterised isolates among laboratories has parallelised research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates identified previously to have aberrant phenotypes varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37{degrees}C, invasion of chicken and human cell lines and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains.\n\nImpact statementIn this paper, we comment on the changing role of laboratory reference strains. While the model organism allows basic comparison within and among laboratories, it is important to remember the effect even small differences in isolate genomes can have on the validity and reproducibility of experimental work. We quantify differences in 23 reference Campylobacter genomes and compare them with observable differences in common laboratory phenotypes.\n\nData summaryShort read data are archived on the NCBI SRA associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467).\n\nAll assembled genomes are also available on FigShare (doi: 10.6084/m9.figshare.7849268). Phylogeny visualised on microreact: https://microreact.org/project/NCTC11168.\n\nRepositoriesShort read data are archived on the NCBI SRA repository, associated with BioProject accession PRJNA517467 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA517467; Table S1).\n\nThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

microbiology↗

bioRxiv2019

Chemical biofilm dislodgement with chelating and reducing agents in comparison to sonication: implications for the diagnosis of implant associated infection

Sonication of removed devices improved the microbiological diagnosis of infection. Recently, chemical agents have been investigated for dislodgement of biofilms, including the chelating agent ethylenediaminetetraacetic acid (EDTA) and the reducing agent dithiothreitol (DTT). We compared the efficacy of chemical methods (EDTA and DTT) to sonication for biofilm dislodgement. Staphylococcus epidermidis (ATCC 35984) and Pseudomonas aeruginosa (ATCC 53278) biofilms were grown on porous glass beads for 3 days. After biofilm formation, beads were exposed to 0.9% saline, sonication and/or chemical agents. Quantitative and qualitative biofilm analyses were performed by colony counting (CFU/ml), isothermal microcalorimetry and scanning electron microscopy. The colony counts after treatment with EDTA and DTT were similar to those after exposure to 0.9% saline (6.3, 6.1 and 6.0 log CFU/ml, respectively) for S. epidermidis biofilms, and (5.1, 5.2 and 5.0 log CFU/ml, respectively) for P. aeruginosa biofilm. Sonication detected higher CFU counts (7.5 log CFU/ml) for S. epidermidis; (p<0.05) and 6.5 log for P. aeruginosa biofilm (p <0.05). Concordant results were detected with isothermal microcalorimetry and scanning electron microscopy. In conclusion, the CFU count after treatment of S. epidermidis or P. aeruginosa biofilms with EDTA and DTT was similar to those observed after 0.9% saline used as control. In contrast, sonication was superior to chemical methods for biofilm dislodgment and detection of microorganisms in sonication fluid. In conclusion, our study showed that sonication is superior to chemical method to dislodge bacterial biofilm from artificial surface and should be considered as standard diagnostic method for biofilm detection in implant-associated infections.

microbiology↗

bioRxiv2019

Metabolic and metagenomic profiling of hydrocarbon-degrading microorganisms obtained from the deep biosphere of the Gulf of Mexico

Marine microbes are capable of degrading hydrocarbons; however, those inhabiting the deep biosphere (>1000 m) remain largely unexplored. Microbial metabolism could lead to the generation of diverse chemistries (some with therapeutic activities), but the impact of using hydrocarbons as the sole source of microbial energy in the synthesis of metabolites, remains obscure. Here, we investigated the metagenomic and metabolomic profile of two deep-marine sediments (>1 200 m deep, designated as A7 and B18) collected from the Gulf of Mexico (GM) when grown for 28 days with a simple mixture of 4 hydrocarbons and complex hydrocarbon mixture (petroleum API 40) as their sole source of energy. State of the art techniques and analysis (e.g., Global Natural Products Social Molecular Networking, network annotation propagation [NAP], and MS2LDA) were used to describe the chemistries associated to the microbial utilization of hydrocarbons. The metagenomic sequencing analysis suggests a predominant abundance of Proteobacteria in environmental and API 40-enriched samples, while the abundance of Pseudomonas increased after microbial growth with API 40. The metabolomic analysis suggests the presence of diverse chemistries predominantly associated with lipid and lipid-like and phenyl propanoids and polyketides superclass (Classyfire annotation). Hydrocarbon derivatives were detected as carboxylic acids (e.g., azelaic and sebacic acid) or alcohols, while non-hydrocarbon related chemistries were also detected including tetracycline-related metabolites and sphinganines. Our study provides valuable chemical and microbiological information of microbes inhabiting one of the most understudied ecosystems in the earth, the deep marine biosphere.

microbiology↗

bioRxiv2019

The Role of fosA in Challenges with Fosfomycin Susceptibility Testing of Multispecies Klebsiella pneumoniae Carbapenemase-Producing Clinical Isolates

With multidrug resistant (MDR) Enterobacteriales on the rise, a non-toxic agent with a unique mechanism of action such as fosfomycin seems attractive. However, establishing accurate fosfomycin susceptibility testing for non-E. coli in a clinical microbiology laboratory remains problematic. We evaluated fosfomycin susceptibility by multiple methods with multiple strains and species of KPC-producing clinical isolates collected at a single center between 2008 and 2016. In addition, we assessed the presence of fosfomycin resistance genes from whole genome sequencing (WGS) data using NCBIs AMRFinder and custom HMM search. Susceptibility testing was performed using glucose-6-phosphate supplemented fosfomycin E-Test and Kirby-Bauer disk diffusion (DD) assays, and compared to agar dilution. Clinical Laboratory and Standards Institute (CLSI) breakpoints for E. coli were applied for interpretation. Overall, 63% (60/96) of isolates were susceptible by E-Test, 70% (67/96) by DD, and 88% (84/96) by agar dilution. FosA was detected in 80% (70/88) of previously sequenced isolates, with species-specific associations and alleles, and fosA-positive isolates were associated with higher MIC distributions. Disk potentiation testing was performed using sodium phosphonoformate to inhibit fosA and showed significant increases in the zone diameter of DD testing for isolates that were fosA-positive compared to fosA-negative. The addition of sodium phosphonoformate (PPF) corrected 10/14 (71%) major errors in categorical agreement with agar dilution. Our results indicate that fosA influences the inaccuracy of susceptibility testing by methods readily available in a clinical laboratory when compared to agar dilution. Further research is needed to determine the impact of fosA on clinical outcomes.

microbiology↗

bioRxiv2019

First report of New Clonal groups ST706 and ST1088 from MDR Klebsiella pneumoniae Mexican strains

Multidrug-resistant Klebsiella pneumoniae Mexican strains were characterized for the identification of endemic and pandemic clonal groups. The aims of this study were to know population structure and to identify endemic clonal groups inside K. pneumoniae Mexican strains isolated from clinical sources. We studied 93 isolated strains from three third level hospitals and one family clinic from Mexico City. Identification of the strains was done by conventional microbiological methods and an automated system (Vitek2(R)). The multidrug-resistant phenotype was confirmed following CLSI recommendations, and the strains were classified as MDR, XDR and PDR. Molecular characterization was done by Multilocus Sequence Typing scheme (rpoB, gapA, mdh, pgi, phoE, infB, y tonB). All strains were isolated from hospitalized patients, the most frequent sources were urine and blood cultures. Population structure of K. pneumoniae was clonal, 30 ST were identified, six of them are commonly found. The Clonal complex ST25, ST36, S5392, ST405 and ST551 were isolated from clinical sources, ST1088 was isolated from surfaces of hospital environment.

microbiology↗

bioRxiv2019

Oregano powder reduces Streptococcus and increases SCFA concentration in a mixed bacterial culture assay of chicken.

Food borne illnesses have a world-wide economic impact and industries are continuously developing technologies to reduce the spread of disease caused by microorganisms. Antimicrobial growth promoters (AGPs) have been used to decrease microbiological infections in animals and their potential transfer to humans. In recent years there has been a global trend to remove AGPs from animal feed in an attempt to reduce the spread of antimicrobial resistant genes into the human population. Phytobiotics, such as oregano powder, are one of the potential replacements for AGPs due to their well-established antimicrobial components. 16S rRNA gene amplicons were used to determine the effect of oregano powder (1% w/v) on the microbiota of mixed bacterial cell cultures, which were obtained from the ceca of traditionally grown meat chickens (broilers). Oregano powder had a mild effect on the microbial cell cultures increasing Enterococcus faecium, rearranging ratios of members in the genus Lactobacillus and significantly reducing the genus Streptococcus (p=1.6e-3). Beneficial short chain fatty acids (SCFA), acetic and butyric acid, were also significantly increased in oregano powder supplemented cultures. These results suggest that oregano powder at a concentration of 1 % (w/v) may have beneficial influences on mixed microbial communities and SCFA production.

microbiology↗

bioRxiv2019

Microbial community shifts associated with the ongoing stony coral tissue loss disease outbreak on the Florida Reef Tract

As many as 22 of the 45 coral species on the Florida Reef Tract are currently affected by stony coral tissue loss disease (SCTLD). The ongoing disease outbreak was first observed in 2014 in Southeast Florida near Miami and as of early 2019 has been documented from the northernmost reaches of the reef tract in Martin County down to Key West. We examined the microbiota associated with disease lesions and apparently healthy tissue on diseased colonies of Montastraea cavernosa, Orbicella faveolata, Diploria labyrinthiformis, and Dichocoenia stokesii. Analysis of differentially abundant taxa between disease lesions and apparently healthy tissue identified five unique amplicon sequence variants enriched in the diseased tissue in three of the coral species, namely an unclassified genus of Flavobacteriales and sequences identified as Fusibacter (Clostridiales), Planktotalea (Rhodobacterales), Algicola (Alteromonadales), and Vibrio (Vibrionales). In addition, several groups of likely opportunistic or saprophytic colonizers such as Epsilonbacteraeota, Patescibacteria, Clostridiales, Bacteroidetes, and Rhodobacterales were also enriched in SCTLD disease lesions. This work represents the first microbiological characterization of SCTLD, as an initial step toward identifying the potential pathogen(s) responsible for SCTLD.

microbiology↗

bioRxiv2019

Fighting antimicrobial resistance in Pseudomonas aeruginosa with machine learning-enabled molecular diagnostics

The growing importance of antibiotic resistance on clinical outcomes and cost of care underscores the need for optimization of current diagnostics. For a number of bacterial species antimicrobial resistance can be unambiguously predicted based on their genome sequence. In this study, we sequenced the genomes and transcriptomes of 414 drug-resistant clinical Pseudomonas aeruginosa isolates. By training machine learning classifiers on information about the presence or absence of genes, their sequence variation, and gene expression profiles, we generated predictive models and identified biomarkers of susceptibility or resistance to four commonly administered antimicrobial drugs. Using these data types alone or in combination resulted in high (0.8-0.9) or very high (>0.9) sensitivity and predictive values, where the relative contribution of the different categories of biomarkers strongly depended on the antibiotic. For all drugs except for ciprofloxacin, gene expression information substantially improved diagnostic performance. Our results pave the way for the development of a molecular resistance profiling tool that reliably predicts antimicrobial susceptibility based on genomic and transcriptomic markers. The implementation of a molecular susceptibility test system in routine clinical microbiology diagnostics holds promise to provide earlier and more detailed information on antibiotic resistance profiles of bacterial pathogens and thus could change how physicians treat bacterial infections.

microbiology↗

bioRxiv2019

Evidence for extensive anaerobic dechlorination and transformation of the pesticide chlordecone (C10Cl10O) in soil from Guadeloupe

Chlordecone (C10Cl10O) is a bishomocubane molecule, that has been used as pesticide, in many countries in Europe, America, and Africa, from the 1960s to 1990s. In the French West Indies, the historic use of chlordecone to control banana weevil infestations has resulted in pollution of large land areas. Although currently banned, chlordecone persists because it adsorbs strongly to soil and its complex structure is stable, particularly under aerobic conditions. A leaching model established that CLD pollution will last in French west indies soils several decades to half a millennium depending on soil type. However, abiotic chemical transformation catalyzed by reduced vitamin B12 has been shown to break down chlordecone by opening the cage structure to produce C9 polychloroindenes, and more recently these C9 polychloroindenes were also observed as products of anaerobic microbiological transformation by Citrobacter. To assess the potential for bioremediation, the anaerobic biotransformation of chlordecone by microbes native to soils from the French West Indies was investigated. Anaerobic microcosms were constructed from chlordecone impacted Guadeloupe soil and sludge to mimic natural attenuation and eletron donor-stimulated reductive dechlorination. Original microcosms and transfers were incubated over a period of 8 years, during which they were repeatedly amended with chlordecone and electron donor (ethanol and acetone). Using LC/MS, chlordecone and degradation products were detected in all the biologically active microcosms. Observed products in active incubations included monohydro-, dihydro- and trihydrochlordecone derivatives (C10Cl10-nO2Hn, n= 1,2,3), as well as \"open cage\" C9 polychloroindene compounds (C9Cl5-nH3+n, n=0,1,2) and C10 carboxylated polychloroindene derivatives (C10Cl4-nO2H4+n, n=0-3). Products with as many as 9 chlorine atoms removed were detected. These products were not observed in sterile incubations. Chlordecone concentrations decreased in active microcosms as concentrations of products increased, indicating that anaerobic dechlorination processes have occurred. An crude estimation of partitioning coefficients between soil and water showed that carboxylated intermediates sorb poorly, and as a consequence may be flushed away while polychlorinated indenes sorb strongly to soil. Microbial community analysis in microcosms showed enrichment of anaerobic fermenting and acetogenic microbes possibly involved in anaerobic chlordecone biotransformation. It thus should be possible to stimuilate anaerobic dechlorination through donor amendment to contaminated soils, particularly as some metabolites (in particular pentachloroindene) were already detected in field samples as a result of intrinsic processes. Extensive dechlorination in the microcosms, with evidence for up to 9 Cl atoms removed from the parent molecule is game-changing, giving hope to the possibility of using bioremediation to reduce the impact of CLD contamination.

microbiology↗

bioRxiv2019

Inactivation of genes in oxidative respiration and iron acquisition pathways in pediatric clinical isolates of small-colony variant Enterobactericeae

Isolation of bacterial small colony variants (SCVs) from clinical specimens is not uncommon and can fundamentally change the outcome of the associated infections. Bacterial SCVs often emerge with their normal colony phenotype (NCV) co-isolates in the same sample. The genetic and biochemical basis of SCV emergence in vivo is not well understood in Gram-negative bacteria. In this study, we interrogated the causal genetic lesions of SCV growth in three pairs of NCV and SCV co-isolates of Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei. We confirmed the isogenic basis of SCV emergence, as there were only 4 single nucleotide variants in SCV for E. coli, 5 in C. freundii, and 8 in E. hormaechei, with respect to their NCV co-isolate. In addition, a 10.2kb chromosomal segment containing 11 genes was deleted in the E. hormaechei SCV isolate. Intriguingly, each SCV had at least one coding change in a gene associated with bacterial oxidative respiration and another involved iron capture. Chemical rescue confirmed the causal role of heme biosynthesis in E. coli and C. freundii and lipoic acid in E. hormaechei SCV isolates. Genetic rescue restored normal growth under aerobic conditions for fes and hemL in C. freundii; hemL in E. coli; and lipA in E. hormaechei SCV isolates. Prototrophic growth in all 3 SCV Enterobacteriaceae species was unaffected under anaerobic culture conditions in vitro, illustrating how SCVs may persist in vivo by abandoning the highly energetic lifestyle in an iron-limiting environment. We propose that the selective loss of functions in oxidative respiration and iron acquisition is indicative of bacterial virulence attenuation for niche specialization and persistence in vivo.\n\nImportance\n\nSmall colony variant (SCV) bacteria are routinely isolated in the clinical microbiology laboratory and can be notoriously difficult to treat. Most studies of the genetic underpinnings of SCV clinical isolates have examined Staphylococcus aureus and few have looked at how SCV emerge in Gram-negative bacteria. Here, we undertook detailed characterization of three clinical isolates of SCV in Escherichia coli, Citrobacter freundii, and Enterobacter hormaechei along with their NCV co-isolates. Genomic sequencing revealed that each SCV had at least one coding change in genes involved in both bacterial oxidative respiration and iron capture. Chemical and genetic rescue revealed that both pathways could be responsible for the small colony variant. Each of the SCV showed no growth defect compared to NCV when incubated under anaerobic conditions, indicating a potential mechanism for SCV survival in vivo. We hypothesize that by retreating to anaerobic environments and avoiding escalating iron competition with the host, SCV have adapted to live to see another day.

microbiology↗

bioRxiv2019

Polyhydroxyalkanoate granule accumulation makes optical density measurement an unreliable method for bacterial growth assessment in Burkholderia thailandensis

Optical density (OD) measurement is the standard method used in microbiology for estimating bacterial concentrations in cultures. However, most studies do not compare these measurements with viable cell counts and assume that they reflect the real cell concentration. Burkholderia thailandensis was recently identified as a polyhydroxyalkanoate (PHA) producer. PHA biosynthesis seems to be coded by an ortholog of the Cupriavidus necator phaC gene. When growing cultures of wildtype strain E264 and an isogenic phaC- mutant, we noted a difference in their OD600 values, although viable cell counts indicated similar growth. Investigating the cellular morphologies of both strains, we found that under our conditions the wildtype strain was full of PHA granules, deforming the cells, while the mutant contained no granules. These factors apparently affected the light scattering, making the OD600 values no longer representative of cell density. We show a direct correlation between OD600 values and the accumulation of PHA. We conclude that OD measurement is unreliable for growth evaluation of B. thailandensis because of PHA production. This study also suggests that B. thailandensis could represent an excellent candidate for PHA bioproduction. Correlation between OD measurements and viable cell counts should be verified on any study realized in B. thailandensis.

microbiology↗