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bioRxiv2022

The Iron Content of Human Serum Albumin Modulates the Susceptibility of Acinetobacter baumannii to Cefiderocol

Mortality rates of patients infected with Acinetobacter baumannii treated with cefiderocol (CFDC) were not as favorable as the best available treatment for pulmonary and bloodstream infections. Previous studies showed that the presence of human serum albumin (HSA) or HSA-containing fluids like human pleural fluid (HPF) or human serum (HS) in the growth medium is correlated with a decrease in the expression of genes associated with high-efficiency iron uptake systems. These observations may explain the less-than-ideal performance of CFDC in pulmonary and bloodstream infections because ferric siderophore transporters enhance penetration of CFDC into the cells cytosol. Removal of HSA from HPF or HS resulted in a reduction of the minimal inhibitory concentration of CFDC. Concomitant with these results, there was an enhancement of the expression of genes associated with high-efficiency iron uptake systems. In addition to inducing modifications in iron-uptake gene expression, removal of HSA also decreased the expression of {beta}-lactam resistance genes. Taken together, these observations indicate that environmental HSA has a role in the expression levels of selected A. baumannii. Furthermore, removal of iron from HSA had the same effect as removal of HSA on the expression of genes associated with high-efficiency iron uptake systems, suggesting that at least one of the mechanisms by which HSA regulates the expression of selected genes is through acting as an iron supplier. IMPORTANCECefiderocol (CFDC) is a new antibiotic that combines its major bactericidal activity, i.e., inhibition of the Gram-negative bacterial cell wall synthesis, with a first in its class mechanism of cell penetration. The siderophore-like moiety facilitates entry through receptors that recognize ferric-siderophore complexes. Recent trials showed that treating pulmonary and bloodstream Acinetobacter baumannii infections with CFDC did not result in the same outcomes as treating other pathogens. Our studies indicated that exposure to human fluids that contain human serum albumin (HSA) increases the MIC values of CFDC. Results described in this work show that HSA is responsible for a reduction in susceptibility of A. baumannii to CFDC. Furthermore, the presence of HSA in the milieu produces a reduction in levels of expression of proteins associated with high-affinity iron uptake systems and enhanced expression of {beta}-lactam resistance-associated genes. Deferration of HSA was accompanied by a loss of the ability to modify these genes expression levels. These results indicate that the microbiological activity of CFDC towards A. baumannii is attenuated in the presence of HSA-containing fluids. This unique insight opens up new avenues of investigation. Understanding this phenomenons molecular mechanism will help define methodologies to increase treatment efficiency.

microbiology↗

bioRxiv2022

Phenotypic Assessment of Clinical Escherichia coli Isolates Predicts Uropathogenic Potential

For women in the United States, urinary tract infections (UTI) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes [~]80% of uncomplicated UTI. To combat this public health issue, it is vital to characterize UPEC strains as well as differentiate them from commensal strains to reduce the overuse of antibiotics. Surprisingly, no genetic signature has been identified which clearly separates UPEC from other E. coli. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and non-pathogenic E. coli strain MG1655 K12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. These data were able to predict the severity of bacterial burden in both the urine and bladders using a well-established experimental mouse model of UTI. Multiple linear regression using three different phenotypic assays, growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted r2=0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted r2=0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. ImportanceUrinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC). These strains can commensally colonize the gut, but upon introduction to the urinary tract, can infect the host and cause disease. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic signature. Here, we have used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to successfully predict uropathogenic potential.

microbiology↗

bioRxiv2022

Melaninization Reduces Cryptococcus neoformans Susceptibility to Mechanical Stress

Melanin is a complex pigment found in various fungal species that is associated with a multitude of protective functions against environmental stresses. In Cryptococcus neoformans, melanin is synthesized from exogenous substrate and deposited in the cell wall. Although melanin is often cited as a protector against mechanical stress, there is a paucity of direct experimental data supporting this claim. To probe whether melanin enhances cellular strength, we used ultrasonic cavitation and French pressure cell press to stress cryptococcal cells and then measured changes in cellular morphology and survival for melanized and non-melanized C. neoformans. Melanized yeast exhibited lower rates of fragmentation and lower decreases in cell area when compared to non-melanized yeast after sonication and French press conditions. Our results indicate that melanization protects against some of the morphologic changes initiated by mechanical energy derived from either sonic cavitation or French press, thus supporting the notion that this pigment provides mechanical strength to fungal cell walls. ImportanceMelanin has been shown from prior experiments in microbiology to be associated with protection against environmental stressors and has often been cited as being associated with mechanical stress protection. However, there is a lack of direct experimentation to confirm this claim. By examining the response of melanized and non-melanized C. neoformans to sonication and French press, we report differences in outcomes dependent not only based on melanization status but also culture age. Such findings have important implications in the design and interpretation of laboratory experiments involving C. neoformans. In addition, uncovering some of melanins mechanical properties promotes further research into fungal melanins applications in healthcare and industry.

microbiology↗

bioRxiv2022

Machine learning identification of Pseudomonas aeruginosa strains from colony image data

When grown on agar surfaces, microbes can produce distinct multicellular spatial structures called colonies, which contain characteristic sizes, shapes, edges, textures, and degrees of opacity and color. For over one hundred years, researchers have used these morphology cues to classify bacteria and guide more targeted treatment of pathogens. Advances in genome sequencing technology have revolutionized our ability to classify bacterial isolates and while genomic methods are in the ascendancy, morphological characterization of bacterial species has made a resurgence due to increased computing capacities and widespread application of machine learning tools. In this paper, we revisit the topic of colony morphotype on the within-species scale and apply concepts from image processing, computer vision, and deep learning to a dataset of 69 environmental and clinical Pseudomonas aeruginosa strains. We find that colony morphology and complexity under common laboratory conditions is a robust, repeatable phenotype on the level of individual strains, and therefore forms a potential basis for strain classification. We then use a deep convolutional neural network approach with a combination of data augmentation and transfer learning to overcome the typical data starvation problem in biological applications of deep learning. Using a train/validation/test split, our results achieve an average validation accuracy of 92.9% and an average test accuracy of 90.7% for the classification of individual strains. These results indicate that bacterial strains have characteristic visual fingerprints that can serve as the basis of classification on a sub-species level. Our work illustrates the potential of image-based classification of bacterial pathogens and highlights the potential to use similar approaches to predict medically relevant strain characteristics like antibiotic resistance and virulence from colony data. Author SummarySince the birth of microbiology, scientists have looked at the patterns of bacterial growth on agar (colony morphology) as a key tool for identifying bacterial species. We return to this traditional approach with modern tools of computer vision and deep learning and show that we can achieve high levels of classification accuracy on a within-species scale, despite what is considered a data-starved dataset. Our results show that strains of the environmental generalist and opportunistic pathogen Pseudomonas aeruginosa have a characteristic morphological fingerprint that enables accurate strain classification via a custom deep convolutional neural network. Our work points to extensions towards predicting phenotypes of interest (e.g. antibiotic resistance, virulence), and suggests that sample size limitations may be less restrictive than previously thought for deep learning applications in biology, given appropriate use of data augmentation and transfer-learning tools.

microbiology↗

bioRxiv2022

In-vitro activity of Cefiderocol against clinical isolates of meropenem-resistant Klebsiella pneumoniae from India.

BackgroundCefiderocol (FDC), a novel siderophore drug, is active against gram negative bacteria producing carbapenemases, including metallo-beta-lactamases. ObjectiveTo compare the in-vitro activity of FDC with ceftazidime-avibactam (CZA), CZA/ aztreonam (AT) combination and colistin (CST), in clinical isolates of meropenem-resistant (MER-R) Klebsiella pneumoniae. MethodsFrom 2052 clinical specimens submitted for culture testing, 245 K. pneumoniae isolates were recovered within a six month period in 2021. 103 non-duplicate, non-outbreak, MER-R (MIC >4 g/ml) strains were included in the study. Identification and susceptibility was performed using VITEK-2 (bioMerieux). 10 meropenem-susceptible isolates served as controls. For FDC, BMD was performed after in-house standardisation. Disc diffusion (Liofilchem, Italy) and broth microdilution (BMD; ComASP, STC, Liofilchem, Italy) were used for susceptibility testing of CZA and CST respectively. Synergy testing for CZA and aztreonam (AT) was performed using disk approximation method. CLSI breakpoints were used for interpretation of results. ResultsFor FDC, MIC50 and MIC90 was 2 g/ml and 8 g/ml, respectively. 80% isolates were susceptible to FDC. 26.2% isolates were susceptible to CZA, synergy testing with CZA/ AT was positive for 74 (72%) of the isolates. 89.3% had intermediate susceptibility to CST. Nine (8.7%) were susceptible only to FDC. ConclusionFDC is active in-vitro against MER-R K. pneumoniae > CZA/AT> CZA > CST, as observed in this study, applying CLSI criteria. Clinico-microbiological studies should be performed for assessing clinical efficacy of this novel drug in this region with high prevalence of carbapenem resistance among gram-negative organisms.

microbiology↗

bioRxiv2022

Long-read powered viral metagenomics in the oligotrophic Sargasso Sea

In the summer months, the waters of the Sargasso Sea are nutrient-limited and strongly stratified, serving as a model system for the predicted warmer and nutrient-limited oceans of the Anthropocene. The dominant microorganisms of surface waters are key drivers of the global carbon cycle. However, the viruses of the Sargasso Sea that shape these host communities and influence host biogeochemical function are not well understood. Here, we apply a hybrid sequencing approach that combines short- and long reads to survey Sargasso Sea phage communities via metagenomics at the viral maximum (80m) and mesopelagic (200m) depths. Taxonomically, we identified 2,301 Sargasso Sea phage populations (~species-level taxonomy) across 186 genera. Over half of the phage populations lacked representation in other global ocean viral metagenomes, whilst 177 phage genera lacked representation in phage isolate databases. Viral fraction and cell-associated viral communities captured in short-read data were distinct and decoupled at both depths, possibly indicating low active lytic viral replication in the Sargasso Sea, with viral turnover occurring across periods longer than the sampling period of three days. Inclusion of long read data was critical for (1) the identification of 79 ecologically important and common viral genomes; (2) capturing the extent of viral genome microdiversity; and (3) enabling the recovery of hypervariable regions in viral genomes predicted to encode proteins involved in host recognition, DNA synthesis and DNA packaging. Host prediction was only possible for ~4% of viral populations. Genomes of phages known to infect Prochlorococcus and Pelagibacter were poorly represented in our data, supporting recent evidence of low infection levels in the dominant bacterial taxa of oligotrophic regions. SubjectsBioinformatics, Genomics, Marine Biology, Microbiology, Virology Sequence data accession numbersPRJNA767318

microbiology↗

bioRxiv2022

Identification of 2-amino benzothiazoles with bactericidal activity against Mycobacterium tuberculosis

We identified an amino-benzothiazole scaffold from a whole cell screen against recombinant Mycobacterium tuberculosis under expressing the essential signal peptidase LepB. The seed molecule had two-fold higher activity against the LepB hypomorph. Through a combination of purchase and chemical synthesis we explored the structure activity relationship for this series; 34 analogs were tested for anti-tubercular activity and for cytotoxicity against eukaryotic cells. We identified molecules with improved potency and reduced cytotoxicity. However, molecules did not appear to target LepB directly and did not inhibit protein secretion. Key compounds showed good permeability, low protein binding, and lack of CYP inhibition, but metabolic stability was poor with short half-lives. The seed molecule showed good bactericidal activity against both replicating and non-replicating bacteria, as well as potency against intracellular M. tuberculosis in murine macrophages. Overall, the microbiological properties of the series are attractive if metabolic stability can be improved, and identification of the target could assist in development of this series.

microbiology↗

bioRxiv2022

Cyclitol secondary metabolism is a central feature of Burkholderia leaf symbionts

The symbioses between plants of the Rubiaceae and Primulaceae families with Burkholderia bacteria represent unique and intimate plant-bacterial relationships. Many of these interactions have been identified through PCR-dependent typing methods, but there is little information available about their functional and ecological roles. We assembled seventeen new endophyte genomes representing endophytes from thirteen plant species, including those of two previously unknown associations. Genomes of leaf endophytes belonging to Burkholderia s.l. show extensive signs of genome reduction, albeit to varying degrees. Except for one endophyte, none of the bacterial symbionts could be isolated on standard microbiological media. Despite their taxonomic diversity, all endophyte genomes contained gene clusters linked to the production of specialized metabolites, including genes linked to cyclitol sugar analog metabolism and in one instance non-ribosomal peptide synthesis. These genes and gene clusters are unique within Burkholderia s.l. and are likely horizontally acquired. We propose that the acquisition of secondary metabolite gene clusters through horizontal gene transfer is a prerequisite for the evolution of a stable association between these endophytes and their hosts.

microbiology↗

bioRxiv2022

Origin and dynamics of Mycobacterium tuberculosis subpopulations that predictably generate drug tolerance and resistance

Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis (Mtb) population. We developed a multi-transwell robotic system to perform numerous parallel cultures of genetically barcoded Mtb exposed to steady-state concentrations of rifampicin to uncover these difficult to eliminate minority populations. We found that tolerance repeatedly emerged from at least two subpopulations of barcoded cells, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed glpK phase variation mutants were associated with up to 16 % of the resistant cultures. Drug tolerance and resistance were eliminated in{Delta} ruvA mutant consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug tolerant subpopulations whose elimination may be critical to developing rapid and resistance free cures. ImportanceTuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter into a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult to eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

microbiology↗

bioRxiv2022

Multidisciplinary approach to the study of artworks: environmental conditions, color palette, microbial colonization and nannofossils in Las Musas painting

Over time, cultural heritage has become a key for comprehending and developing our society at an individual and group level, as it provides fundamental information about our origins, specific temporary space, materials availability using current technology, artists intention, and site weather conditions. Here, we investigated the physical characteristics of an Italian large-format artwork diptych, located on the ceiling of the National Theater of Costa Rica, to evaluate its pictorial palette as well as the tropical climatological conditions and the fungal aerial spore concentration. We characterized the role of two innovative software tools, as they have direct connection with an effective microbiological sampling and description of secrets encompassed in each pictorial layer during the process of creation and intention. We further found that eight genera of calcareous nannofossils could be associated with the optical properties of the artwork and the effects that the artist wanted to portray through his creative process.

microbiology↗

bioRxiv2022

Development of a polymicrobial checkerboard assay as a tool for determining combinatorial antibiotic effectiveness in polymicrobial communities

AIMSTo establish a methodology for identifying the effects of combinatorial antibiotic treatment on individual members of a polymicrobial community. METHODS AND RESULTSBoth gentamicin and ceftazidime were diluted to concentrations ranging from 0.06 g ml-1 to 128 g ml-1. An equal ratio polymicrobial community of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, and Acinetobacter baumannii was inoculated into the combined antibiotics in the checkerboard and incubated for 18 hours in static conditions. After incubation, visible turbidity of the overall community was recorded, and bacteria from the wells were diluted to 10-3 and then spot plated on selective and differential media. After 24 hours, colony-forming unit (CFU) counts were obtained for each species. CONCLUSIONSVisible turbidity is not truly indicative of cell viability, and the polymicrobial community can decrease the antibiotic susceptibility of P. aeruginosa, rendering the clinically-established beneficial combination of gentamicin and ceftazidime ineffective. SIGNIFICANCEPrevious checkerboard methodology which focuses on using visible turbidity to determine monomicrobial antibiotic susceptibility fails to account for polymicrobial cooperation that has been shown to reduce antibiotic efficacy. Our new methodology could be implemented in clinical microbiology laboratories with minimal impact on the overall time for diagnosis.

microbiology↗

bioRxiv2022

Genomic epidemiology of Corynebacterium diphtheriae in New Caledonia

ObjectivesAn increasing number of isolations of Corynebacterium diphtheriae has been observed in recent years in the archipelago of New Caledonia. We aimed to analyze the clinical and microbiological features of samples with C. diphtheriae. MethodsAll C. diphtheriae isolates identified in New Caledonia from May 2015 to May 2019 were included. For each case, a retrospective consultation of the patient files was conducted. Antimicrobial susceptibility phenotypes, tox gene and diphtheria toxin expression, biovar and the genomic sequence were determined. Core genome multilocus sequence typing (cgMLST), 7-gene MLST and search of genes of interest were performed from genomic assemblies. Results58 isolates were included, with a median age of patients of 28 years (range: 9 days to 78 years). Cutaneous origin accounted for 51 of 58 (87.9%) isolates, and C. diphtheriae was associated with Staphylococcus aureus and/or Streptococcus pyogenes in three quarters of cases. Half of cases came either from the main city Noumea (24%, 14/58) or from the sparsely populated island of Lifou (26%, 15/58). Six tox-positive isolates were identified, associated with recent travel to Vanuatu; 5 of these cases were linked and cgMLST confirmed recent transmission. Two cases of endocarditis in young female patients with a history of rheumatic fever involved tox-negative isolates. The 58 isolates were mostly susceptible to commonly used antibiotics. In particular, no isolate was resistant to the first-line molecules amoxicillin or erythromycin. Resistance to tetracycline was found in a genomic cluster of 17 (29%) isolates, 16 of which carried the tetO gene. There were 13 cgMLST sublineages, most of which were also observed in the neighboring country Australia. ConclusionsCutaneous infections may harbor non-toxigenic C. diphtheriae isolates, which circulate largely silently in non-specific wounds. The possible introduction of tox-positive strains from a neighboring island illustrates that diphtheria surveillance should be maintained in New Caledonia, and that immunization in neighboring islands must be improved. Genomic sequencing uncovers how genotypes circulate locally and across neighboring countries.

microbiology↗

bioRxiv2022

Cell-density regulated adhesins contribute to early disease development and adhesion in Ralstonia solanacearum

Adhesins (adhesive proteins) help bacteria stick to and colonize diverse surfaces and often contribute to virulence. The genome of the bacterial wilt pathogen Ralstonia solanacearum (Rs) encodes dozens of putative adhesins, some of which are upregulated during plant pathogenesis. Little is known about the role of these proteins in bacterial wilt disease. During tomato colonization, three putative Rs adhesin genes were upregulated in a {Delta}phcA quorum sensing mutant that cannot respond to high cell densities: radA (Ralstonia adhesin), rcpA (Ralstonia collagen-like protein), and rcpB. Based on this differential gene expression, we hypothesized that adhesins repressed by PhcA contribute to early disease stages when Rs experiences a low cell density. During root colonization Rs upregulated rcpA and rcpB, but not radA, relative to bacteria in the stem at mid-disease. Root attachment assays and confocal microscopy with {Delta}rcpA/B and {Delta}radA revealed that all three adhesins help Rs attach to tomato seedling roots. Biofilm assays on abiotic surfaces found that Rs does not require RadA, RcpA, or RcpB for interbacterial attachment (cohesion), but these proteins are essential for anchoring aggregates to a surface (adhesion). However, Rs did not require the adhesins for later disease stages in planta, including colonization of the root endosphere and stems. Interestingly, all three adhesins were essential for full competitive fitness in planta. Together, these infection stage-specific assays identified three proteins that contribute to adhesion and the critical first host-pathogen interaction in bacterial wilt disease. ImportanceEvery microbe must balance its need to attach to surfaces with the biological imperative to move and spread. The high-impact plant pathogenic bacterium Ralstonia solanacearum can stick to biotic and abiotic substrates, presumably using some of the dozens of putative adhesins encoded in its genome. We confirmed the functions and identified the biological roles of several afimbrial adhesins. By assaying the competitive fitness and the success of adhesin mutants in three individual plant compartments, we identified the specific disease stages and host tissues where three previously cryptic adhesins contribute to bacterial success. Combined with tissue-specific regulatory data, this work indicates that R. solanacearum deploys distinct adhesins that help it succeed at different stages of plant pathogenesis. Research AreasPlant Microbiology, Host-Microbial Interactions, Microbial Pathogenesis

microbiology↗

bioRxiv2022

Rapid Phenotypic Detection of Carbapenem-Resistant E. coli with Fluorogenic Culture Media

Faster, and simpler methods that determine the susceptibility of bacterial pathogens to antibiotics are needed to enable more effective treatment of bacterial infections and reduce the overuse of antibiotics. Here, we demonstrate a simple method for rapidly detecting bacteria and simultaneously determining their antibiotic susceptibility status. We demonstrate the method with E. coli, a high-impact pathogen, and meropenem, a member of the carbapenem class of antibiotics which is usually reserved for multi-drug resistant infections. Inclusion of a fluorogenic oligonucleotide substrate for endonuclease I, a well-conserved nuclease of the Enterobacteriaceae family, in a tryptic soy agar-based media enabled fluorescence-based detection of E. coli growth within 9 hours. Plating established carbapenem-resistant and -susceptible E. coli strains on this media with and without inclusion of meropenem, followed by fluorescent imaging yielded a clear phenotypic measure of the antibiotic susceptibility status of the strains in 9 hours. In addition to its simplicity and fast turnaround time, advantages of this approach include its ability to provide a measure of the bacterial load of the tested samples, and its easy integration into current microbiology laboratory workflows.

microbiology↗

bioRxiv2022

Field testing of an enzymatic quorum quencher coating additive to reduce biocorrosion of steel

Microbial colonization can be detrimental to the integrity of metal surfaces and lead to microbiologically influenced corrosion (MIC). Biocorrosion is a serious problem for aquatic and marine industries in the world and severely affects the maritime transportation industry by destroying port infrastructure and increasing fuel usage and the time and cost required for maintenance of transport vessels. Here, we evaluate the potential of a stable quorum quenching lactonase enzyme to reduce biocorrosion in the field. Over the course of 21 months, steel samples coated with lactonase-containing acrylic paint were submerged at two different sites and depths in the Duluth Superior Harbor (DSH; Lake Superior, MN, USA) and benchmarked against controls, including the biological biocide surfactin. In this experiment, the lactonase treatment outperformed the surfactin biocide treatment and significantly reduced the number of corrosion tubercles (37% (p<0.01)) and the corroded surface area (39% (p<0.01)) as compared to the acrylic-coated control coupons. In an attempt to evaluate the effects of signal disruption of surface microbial communities and the reasons for lower corrosion levels, 16S rRNA sequencing was performed and community populations were analyzed. Interestingly, surface communities were similar between all treatments, and only minor changes could be observed. Among these changes, several groups including sulfate-reducing bacteria (SRBs) appeared to correlate with corrosion levels, and more specifically, SRBs abundance levels were lower on lactonase treated steel coupons. We surmise that these minute community changes may have large impacts on corrosion rates. Overall, these results highlight the potential use of stable quorum quenching lactonases as an eco-friendly antifouling coating additive.

microbiology↗

bioRxiv2022

Investigation on the Effectiveness of Lactobacillus plantarum in Enhancing the Folate content of Injera made with Different Cereals

Injera is an Ethiopian fermented pancake-like flatbread made from different cereals. The aim of the study was to investigate the effect of injera making process using different cereals (tef, sorghum, wheat and barley) on folate content and to evaluate the effectiveness of Lactobacillus plantarum (L. plantarum) in enhancing folate of injera made with different cereals. Cereals were used alone or in combination (tef and sorghum (1:1), wheat and sorghum (3:1), sorghum (100 %) and Barley (100 %). L. plantarum previously isolated from tef dough and ersho were used as starters. Folate content of flour, dough and injera was determined by microbiological assay. The contribution of consumption of injera made with different cereals to the folate requirement of children and women of reproductive age was evaluated. Acceptability of injera was estimated by 30 adult healthy volunteers using a 9-point hedonic scale. Among the studied cereals, the highest average folate content (49.9 g/100 g) was observed in 100 % sorghum flour and the least (32.2 g/100 g) in 100 % barley flour, on dry weight basis. After fermentation, the highest average folate content (60.1 g/100 g) was observed in 100 % sorghum dough fermented with L. plantarum and the least (27.6 g/100 g) was observed in the same dough but fermented with ersho. The average folate contents (fresh weight basis) of 100 % sorghum, wheat & sorghum (3:1), tef & sorghum (1:1) and 100 % barley injeras fermented with L. plantarum were 14.48 g/100 g, 15.45 g/100 g, 13.23 g/100 g and 13.13 g/100 g, respectively. Consumption of L. plantarum fermented injera made with different cereals can contribute up to 8 % of the recommended folate intake of women of reproductive age. Injera made with tef and sorghum blend (1:1) and fermented with L. plantarum was highly accepted.

microbiology↗

bioRxiv2022

A la carte, Streptococcus pneumoniae capsular typing: using MALDI-TOF mass spectrometry and machine learning algorithms as complementary tools for the determination of PCV13 serotypes and the most prevalent NON PCV13 serotypes according to Argentina's epidemiology.

Laboratory surveillance of Streptococcus pneumoniae serotypes is crucial for the successful implementation of vaccines to prevent invasive pneumococcal diseases. The reference method of serotyping is the Quellung reaction, which is labor-intensive and expensive. In the last few years, the introduction of MALDI-TOF MS into the microbiology laboratory has been revolutionary. In brief, this new technology compares protein profiles by generating spectra based on the mass to charge ratio (m/z). We evaluated the performance of MALDI-TOF MS for typing serotypes of S. pneumoniae isolates included in the PCV13 vaccine using a machine learning approach. We challenged our classification algorithms in "real time" with a total of new 100 isolates of S. pneumoniae from Argentinian nationwide surveillance. Our best approach could correctly identify the isolates with a sensitivity of 58.33 % ([95%IC 40.7-71.7]); specificity of 81.48 % ([95%IC 53.6-79.7]); accuracy of 63.0% ([95%IC 61.9-93.7]); PPV of 80.77% ([95%IC 64.5-90.6]) and NPV of 59.46% ([95%IC 48.9-69.2]). In this work, it was possible to demonstrate that the combination of MALDI-TOF mass spectrometry and multivariate analysis allows the development of new strategies for the identification and characterization of Spn isolates of clinical importance; and we consider that by using AI, as more data becomes available the models will get better and more precise.

microbiology↗

bioRxiv2022

Pre-clinical in vitro and in vivo characterization of a maternal vaccination before conception to protect against severe neonatal infections caused by Escherichia coli K1

Preterm birth remains the leading cause of neonatal morbidity and mortality today. Genetic, immunological, and infectious substrates are suspected. Preterm infants are at higher risk of severe neonatal infections and the main cause of bacterial infection in this population is Escherichia coli K1. Unfortunately, women with history of preterm birth have a high risk of recurrence. Therefore, these women constitute a target population for a vaccine, to date non-existent, against E. coli K1 to prevent these infections. In this study, we characterize the immunological and microbiological properties in adult female mice of a live attenuated vaccine candidate and the protection it conferred to newborn mice against severe infection caused by E. coli K1. We show that our E. coli K1 {Delta}aroA vaccine induces a strong immunity driven by polyclonal bactericidal antibodies. In our model of meningitis, pups born from mothers immunized before conception were strongly protected against different strains of E. coli K1 both in early-onset and late-onset diseases. Given the very high rate of mortality and neurological sequalae in neonatal meningitis caused by E. coli K1, this pre-clinical study provides a proof-of-concept for the development of a vaccine strategy against E. coli K1 severe infection in women at risk of preterm birth.

microbiology↗