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La Trobe University Faculty of Science, Technology and Engineering Murray Darling Freshwater Research CentreMDFRC item.Routine microscopic examination of biomass samples from many activated sludge plants around the world reveals the presence of large numbers of both Gram positive and Gram negative coccoid cells often arranged in distinctive tetrads or sheets of tetrads. Three Gram negative tetrad cocci were successfully isolated into pure culture using micromanipulation from samples of biomass from plants in Tamworth, N.S.W., Australia, Verona, Italy and Macau. After extensive phenotypic characterisation and 16S rDNA sequence analysis, all isolates showed high overall similarity to each other, but were phylogenetically quite different to any previously sequenced bacterium. They have therefore been classified as three species of a new genus, the genus Amaricoccus in the a Proteobacteria in the domain Bacteria. None could accumulate polyphosphate granules, and production of intracellular poly-beta-hydroxybutyrate (PHB) granules depended on the carbon source used in the medium. A similar characterisation of the original isolate of the G-bacteria described by Cech and Hartman showed this organism also belonged to the same genus as the other three isolates, but as a distinct species. The practical implications of these observations are discussed.

In the current study, the optimization of ectoine production by Nesterenkonia xinjiangensis and purification of ectoine from the bacterial cell extract were performed for the first time. Various carbon sources (glucose, sucrose, maltose, lactose, mannitol, and xylose) and nitrogen sources (ammonium nitrate, ammonium phosphate, ammonium chloride, ammonium oxalate, ammonium sulphate, and ammonium acetate), were used to optimize ectoine production. Subsequently, the effects of salt, pH and, concentrations of carbon and nitrogen source on ectoine production were optimized by response surface methodology (RSM). Ultimately, high pure (over 99%) and yield (98%) of ectoine from bacterial cells extracted was obtained by a single-step process using cation exchange chromatography. This study provides information that higher ectoine production can be achieved from this bacterial isolate by optimizing the factors influencing ectoine production and thus can be used as a new and alternative ectoine producer. © 2023, BAP- FEF.20.001, This study was supported by Agri Ibrahim Cecen University, Scientific Researches Project (Project number: BAP- FEF.20.001).

Summary Members of the genus Pseudomonas inhabit diverse environments, such as soil, water, plants and humans. The variability of habitats is reflected in the diversity of the structure and composition of their genomes. This cosmopolitan bacterial genus includes species of biotechnological, medical and environmental importance. In this study, we report on the most relevant genomic characteristics of Pseudomonas sp. strain ABC1, a siderophore‐producing fluorescent strain recently isolated from soil. Phylogenomic analyses revealed that this strain corresponds to a novel species forming a sister clade of the recently proposed Pseudomonas kirkiae. The genomic information reveals an overrepresented repertoire of mechanisms to hoard iron when compared to related strains, including a high representation of fecI‐fecR family genes related to iron regulation and acquisition. The genome of the Pseudomonas sp. ABC1 contains the genes for non‐ribosomal peptide synthetases (NRPSs) of a novel putative Azotobacter‐related pyoverdine‐type siderophore, a yersiniabactin‐type siderophore and an antimicrobial betalactone; the last two are found only in a limited number of Pseudomonas genomes. Strain ABC1 can produce siderophores in a low‐cost medium, and the supernatants from cultures of this strain promote plant growth, highlighting their biotechnological potential as a sustainable industrial microorganism., ‘Pseudomonas chilensis’ strain ABC1 was fully sequenced. About 5% of the ABC1 genome is devoted to iron acquisition, including 37 ECF sigma factors. Strain ABC1 produces pyoverdine and has the genes for the production of yersiniabactin.

Background Recently it was shown that production of recombinant proteins in E. coli BL21(DE3) using pET based expression vectors leads to metabolic stress comparable to a carbon overfeeding response. Opposite to original expectations generation of energy as well as catabolic provision of precursor metabolites were excluded as limiting factors for growth and protein production. On the contrary, accumulation of ATP and precursor metabolites revealed their ample formation but insufficient withdrawal as a result of protein production mediated constraints in anabolic pathways. Thus, not limitation but excess of energy and precursor metabolites were identified as being connected to the protein production associated metabolic burden. Results Here we show that the protein production associated accumulation of energy and catabolic precursor metabolites is not unique to E. coli BL21(DE3) but also occurs in E. coli K12. Most notably, it was demonstrated that the IPTG-induced production of hFGF-2 using a tac-promoter based expression vector in the E. coli K12 strain TG1 was leading to persistent accumulation of key regulatory molecules such as ATP, fructose-1,6-bisphosphate and pyruvate. Conclusions Excessive energy generation, respectively, accumulation of ATP during recombinant protein production is not unique to the BL21(DE3)/T7 promoter based expression system but also observed in the E. coli K12 strain TG1 using another promoter/vector combination. These findings confirm that energy is not a limiting factor for recombinant protein production. Moreover, the data also show that an accelerated glycolytic pathway flux aggravates the protein production associated “metabolic burden”. Under conditions of compromised anabolic capacities cells are not able to reorganize their metabolic enzyme repertoire as required for reduced carbon processing.

Background Tuberculosis (TB) diagnosis in human immunodeficiency virus (HIV) positive persons is difficult, particularly in resource-limited settings. The relationship between TB culture status and mortality in HIV-positive persons treated for TB is unclear. Methods We evaluated HIV-positive adults treated for TB at or after their first HIV clinic visit in Argentina, Brazil, Chile, Honduras, Mexico or Peru from 2000 to 2015. Anti-tuberculosis treatment included 2 months of isoniazid, rifampicin (RMP)/rifabutin (RBT), pyrazinamide ± ethambutol, followed by continuation phase treatment with isoniazid + RMP/RBT. Results Of 759 TB-HIV patients, 238 (31%) were culture-negative, 228 (30%) had unknown culture status or did not undergo culture and 293 (39%) were culture-positive. The median CD4 at TB diagnosis was 96 (interquartile range 40-228); 636 (84%) received concurrent antiretroviral therapy (ART) and anti-tuberculosis treatment. There were 123 (16%) deaths: 90/466 (19%) with TB culture-negative, unknown or not performed vs. 33/293 (11%) who were TB culture-positive (P = 0.005). In Kaplan-Meier analysis, mortality in TB patients without culture-confirmed disease was higher (P = 0.002). In a Cox model adjusted for age, sex, CD4, ART timing, disease site and stratified by study site, mortality in persons without culture-confirmed TB was not significantly increased compared to those with culture-positive TB (hazard ratio 1.39, 95%CI 0.89-2.16, P = 0.15). Conclusion Most HIV-positive patients treated for TB did not have culture-confirmed TB, and mortality tended to be higher in patients without culture-confirmed disease, although the association was not statistically different after adjusting for other variables. Accurate TB diagnosis in HIV-positive persons is crucial.

Background/aim: Infective endocarditis (IE) is still a significant cause of morbidity and mortality among cardiovascular diseases. ENDOCARDITIS-TR study aims to evaluate the compliance of the diagnostic and therapeutic methods being used in Turkey with current guidelines. Materials and methods: The ENDOCARDITIS-TR trial is a multicentre, prospective, observational study consisting of patients admitted to tertiary centres with a definite diagnose of IE. In addition to the demographic, clinical, microbiological, and echocardiographic findings of the patients, adverse events, indications for surgery, and in-hospital mortality were recorded during a 2-year time interval. Results: A total of 208 IE patients from 7 tertiary centres in Turkey were enrolled in the study. The study population included 125 (60.1%) native valve IE (NVE), 65 (31.3%) prosthetic IE (PVIE), and 18 (8.7%) intracardiac device-related IE (CDRIE). One hundred thirty-five patients (64.9%) were culture positive, and the most frequent pathogenic agent was methicillin-susceptible Staphylococcus aureus (MSSA) (18.3%). Among 155 (74.5%) patients with an indication for surgery, only 87 (56.1%) patients underwent surgery. The all-cause mortality rate was 29.3% in-hospital follow-up. Multivariable Cox regression analysis revealed that absence of surgery when indicated (HR: 3.29 95% CI: 0.93–11.64 p = 0.05), albumin level at admission (HR: 0.46 95% CI: 0.29–0.73 P < 0.01), abscess formation (HR: 2.11 95% CI: 1.01–4.38 p = 0.04) and systemic embolism (HR: 1.78 95% CI: 1.05–3.02 p = 0.03) were ascertained independent predictors of in-hospital all-cause mortality. Conclusion: The short-term results of the ENDOCARDITIS-TR trial showed the high frequency of staphylococcal IE, relatively high in-hospital mortality rates, shortage of surgical treatment despite guideline-based surgical indications and low usage of novel imaging techniques. The results of this study will provide a better insight to physicians in respect to their adherence to clinical practice guidelines. © TÜBİTAK.

Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms . Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum , were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum , whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation . Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.

El presente artículo reporta un caso clínico de otomicosis por Aspergillus en un Cocker Spaniel Inglés macho de 8 años, con diagnóstico previo de anemia hemolítica inmunomediada. El paciente fue atendido por presentar dolor e incomodidad en ambos oídos. Se realizaron análisis sanguíneos, además de citología y cultivo microbiológico de secreción ótica. El Aspergillus niger junto con Malassezia pachydermatis fueron los agentes aislados, para lo cual se instauró una terapia antifúngica oral y tópica con resultados favorables después de 30 días de tratamiento. El moho aislado presentó algunos factores de virulencia importantes como actividad hemolítica, lipasa y amilasa, además de susceptibilidad in vitro a antifúngicos del tipo azoles. En pacientes inmunosuprimidos que presenten otitis externa es necesario realizar cultivos microbiológicos para hongos ambientales, además de evaluar a nivel fenotípico la característica patógena, lo cual ayude a establecer un correcto abordaje terapéutico y monitoreo de enzimas hepáticas por posibles efectos adversos.

Bu çalışma, 2019 yılında Bodrum[Türkiye]'da düzenlenen TRS Kongresi'nde bildiri olarak sunulmuştur. Introduction: Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a well-established diagnostic tool for lung cancer, sarcoidosis, and suspected metastatic extra-thoracic malignancy (ETM). Patients with primary ETM often have hypermetabolic mediastinal/hilar lymph node enlargement in the PET-scan done for initial staging or post treatment follow-up. We aimed to determine the diagnostic performance of EBUS-TBNA and the relationship between PET-SUV values and diagnosis of malignancy metastasis in patients with ETM. Materials and Methods: Results of EBUS-TBNA in ETM patients with suspected MLN metastasis were retrospectively analysed (May 2016 to July 2019). Non-malign results were confirmed for surgery or clinical/radiological follow-up. Lymph nodes with a high FDG-uptake (SUV > 2.5, MLN) were reported as suspicious for metastasis. Results: Of the 588 EBUS procedures, 109 were included in the analysis. Patient' mean age was 62.5 +/- 10.1 years; there were 35 men and 74 women. Primary malignancies were breast cancer in 33, gastrointestinal in 23, female genital tract in 17, head and neck in 14, genitourinary cancer in 13, malignant melanoma in 6, sarcoma in 2 and kaposi sarcoma in 1. According to EBUS-TBNA smear and cell block histopathologic evaluations, 16 patients' results (14.7%) were malignant compatible with metastasis of ETM. Among the 93 patients with non-malignant diagnosis, EBUS-TBNA revealed a granulomatous lympadenitis compatible with sarcoid reaction in 7 and tuberculosis in 2. A total of 9 patients underwent surgical procedures after EBUS-TBNA, with a definitive histological diagnosis of granulomatous lymphadenitis in 2, malignancy in 5 and, reactive lymph node in 2. Overall sensitivity, specificity, positive predictive value, negative predictive value and diagnostic accuracy of EBUS-TBNA were 76.19% (95% CI 52.83-91.78), 100% (95% CI 95.89-100.00), 100%, 94.62% (95% CI 89.12-97.12) and 95.4%, respectively. Conclusion: EBUS-TBNA sampling has high diagnostic performance. Histopathological confirmation requirement for MLN should be kept in mind in patients with ETM, even they have negative EBUS results. Giriş: Endobronşial ultrason-kılavuzluğunda transbronşial iğne aspirasyonu (EBUS-TBİA), akciğer kanseri, sarkoidoz ve metastatik ekstra-torasik malignitelerde (ETM) iyi tanımlanmış bir tanı yöntemidir. Primer ETM evrelemesi ve tedavi sonrası takipte, mediastinal/ hiler lenf nodu (MLN) genişlemesi sık görülmektedir. ETM tanısı olan hastalarda EBUS-TBİA tanı performansı, PET-SUV değerleri ile malign metastaz tanısı arasındaki ilişkiyi araştırmayı hedefledik. Materyal ve Metod: Şüpheli malign metastaz olan ETM hastalarında yapılan EBUS-TBİA sonuçları retrospektif olarak analiz edildi (Mayıs 2016-Temmuz 2019). Malign olmayan sonuçlar cerrahi veya klinik/radyolojik takip ile konfirme edildi. Yüksek FDG-uptake olan (SUV > 2.5, MLN) lenf nodları metastaz şüpheli olarak raporlandı. Bulgular: Toplam 588 EBUS işleminden 109'u analize dahil edildi. Hastaların ortalama yaşı 62.5 ± 10.1; 35'i erkek, 74'ü kadın idi. Primer malignitelerin, 33’ü meme kanseri, 23'ü gastrointestinal, 17'si kadın genital sistem, 14'ü baş-boyun, 13’ü genitoüriner kanser, 6'sı malign melanom, 2'si sarkom ve 1'i kaposi sarkomu idi. EBUS-TBİA yayma ve hücre bloğu histopatolojik sonuçlarına göre 16 hastada (%14.7) ETM metastazı ile uyumlu malignite saptandı. Malign olmayan 93 hastadan 7'sinde sarkoid reaksiyon ile uyumlu granülomatöz lenfadenit, 2'sinde tüberküloz, 5'inde malignite bulundu. EBUS-TBİA sonrası cerrahi ile toplam 9 hastanın 2'sinde granülomatöz lenfadenit, 5'inde malignite ve 2’sinde reaktif lenf nodu saptandı. EBUS-TBİA için toplam duyarlılık, özgüllük, pozitif prediktif değer, negatif prediktif değer ve tanı doğruluğu, sırasıyla %76.19 (%95 GA 52.83-91.78), %100 (%95 GA 95.89-100.00), %100, %94.62 (%95 GA 89.12-97.12) ve %95.4 olarak saptandı. Sonuç: EBUS-TBİA örneklemesi tanı performansı yüksek bir yöntemdir. Negatif EBUS sonuçları olsa da, MLN genişlemesi olan ETM tanılı olgularda histopatolojik doğrulama gerekliliği akılda tutulmalıdır.

In patients with acne, clinical characteristics and anamnesis determine the tests to be ordered. The aetiology of acne is multifactorial. Both sex hormones and metabolic hormones may play a role in the development and severity of acne. From the hormones that may be associated with the aetiopathogenesis of acne, androgens [testosterone, dehydrotestosterone (DHT), and dehydroepiandrosterone sulphate (DHEAS)] are the hormones that influence sebum production most. Although there are not any comorbid endocrinologic diseases in most of the patients with acne, detection of any endocrinopathy is important. An endocrinologic examination is required in female patients with acne in the presence of any characteristics suggesting hyperandrogenism. The necessity of specific, non-standard culture for P. acnes prevents performing routine cultures. One particular case where microbiological sampling is useful in patients with acne is when gram-negative folliculitis is suspected. Cutaneous staphylococcal infections should be considered for differential diagnosis especially in acute lesions and cultures should be taken. © Telif Hakkı 2020 Deri ve Zührevi Hastalıklar Derneği Türkderm - Türk Deri Hastalıkları ve Frengi Arşivi, Galenos Yayınevi tarafından basılmıştır.

Bartonella bacilliformis is the biological agent of Carrion’s disease, a vector-borne, life-threatening human bartonellosis restricted to South America. Here, we report the complete genome sequence of B. bacilliformis KC584 (ATCC 35686). Although it is commonly used as a reference strain, to date, its complete genome has not been published.

When it comes to the discovery and analysis of yet uncharted bacterial traits, pure cultures are essential as only these allow detailed morphological and physiological characterization as well as genetic manipulation. However, microbiologists are struggling to isolate and maintain the majority of bacterial strains, as mimicking their native environmental niches adequately can be a challenging task. Here, we report the diversity-driven cultivation, characterization and genome sequencing of 79 bacterial strains from all major taxonomic clades of the conspicuous bacterial phylum Planctomycetes. The samples were derived from different aquatic environments but close relatives could be isolated from geographically distinct regions and structurally diverse habitats, implying that ‘everything is everywhere’. With the discovery of lateral budding in ‘Kolteria novifilia’ and the capability of the members of the Saltatorellus clade to divide by binary fission as well as budding, we identified previously unknown modes of bacterial cell division. Alongside unobserved aspects of cell signalling and small-molecule production, our findings demonstrate that exploration beyond the well-established model organisms has the potential to increase our knowledge of bacterial diversity. We illustrate how ‘microbial dark matter’ can be accessed by cultivation techniques, expanding the organismic background for small-molecule research and drug-target detection., This work was funded by the Deutsche Forschungsgemeinschaft (grant no. JO 893/4-1) and the Volkswagen foundation (experiment no. 89256). M.Y.G. was funded by the NIH IRP at the US National Library of Medicine. Work in the Mascher lab was supported by the Deutsche Forschungsgemeinschaft (grant no. MA2837/2-2) and the Bundeministerium für Bildung und Forschung in the framework of the ERAnet Synthetic Biology (project: ERASynBio2-ECFexpress). R.A. and A.M. were funded by the Max Planck Society.

Microcystins (MCs) are hepatotoxic and potentially carcinogenic cyanotoxins. They exhibit high structural variability, with nearly 250 variants described to date. This variability can result in incomplete detection of MC variants during lake surveys due to the frequent use of targeted analytical methods and a lack of standards available for identification and quantitation. In this study, Lake Uluabat in Turkey was sampled during the summer of 2015. Phylogenetic analysis of the environmental mcyA sequences suggested Microcystis spp. were the major MC contributors. A combination of liquid chromatography-tandem mass spectrometry (LC-MS/MS), liquid chromatography with UV detection and mass spectrometry (LC-UV-MS), and a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, together with thiol and periodate reactivity, revealed more than 36 MC variants in the lake samples and a strain of M. aeruginosa (AQUAMEB-24) isolated from Lake Uluabat. Only MCs containing arginine at position-4 were detected in the culture, while MC-LA, -LY, -LW and -LF were also detected in the lake samples, suggesting the presence of other MC producers in the lake. The previously unreported MCs MC-(H2)YR (dihydrotyrosine at position-2) (17), [epoxyAdda(5)]MC-LR, [DMAdda(5)]MC-RR (1) and [Mser(7)]MC-RR (8) were detected in the culture and/or field samples. This study is a good example of how commonly used targeted LC-MS methods can underestimate the diversity of MCs in freshwater lakes and cyanobacteria cultures and how untargeted LC-MS methods can be used to comprehensively assess MC diversity present in a new system.

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Some well-known Clostridiales species such as Clostridium difficile and C. perfringens are agents of high impact diseases worldwide. Nevertheless, other foreseen Clostridiales species have recently emerged such as Clostridium tertium and C. paraputrificum. Three fecal isolates were identified as Clostridium tertium (Gcol.A2 and Gcol.A43) and C. paraputrificum (Gcol.A11) during public health screening for C. difficile infections in Colombia. C. paraputrificum genomes were highly diverse and contained large numbers of accessory genes. Genetic diversity and accessory gene percentage were lower among the C. tertium genomes than in the C. paraputrificum genomes. C. difficile tcdA and tcdB toxins encoding homologous sequences and other potential virulence factors were also identified. EndoA interferase, a toxic component of the type II toxin-antitoxin system, was found among the C. tertium genomes. toxA was the only toxin encoding gene detected in Gcol.A43, the Colombian isolate with an experimentally-determined high cytotoxic effect. Gcol.A2 and Gcol.A43 had higher sporulation efficiencies than Gcol.A11 (84.5%, 83.8% and 57.0%, respectively), as supported by the greater number of proteins associated with sporulation pathways in the C. tertium genomes compared with the C. paraputrificum genomes (33.3 and 28.4 on average, respectively). This work allowed complete genome description of two clostridiales species revealing high levels of intra-taxa diversity, accessory genomes containing virulence-factors encoding genes (especially in C. paraputrificum), with proteins involved in sporulation processes more highly represented in C. tertium. These finding suggest the need to advance in the study of those species with potential importance at public health level. © 2019, © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor and Francis Group.

The aim of our study was to characterize the etiology of prosthetic joint infections (PJIs)&mdash, including multidrug-resistant organisms (MDRO)&mdash, by category of infection. A multicenter study of 2544 patients with PJIs was performed. We analyzed the causative microorganisms according to the Tsukayama&rsquo, s scheme (early postoperative, late chronic, and acute hematogenous infections (EPI, LCI, AHI) and &ldquo, positive intraoperative cultures&rdquo, (PIC)). Non-hematogenous PJIs were also evaluated according to time since surgery: &lt, 1 month, 2&ndash, 3 months, 4&ndash, 12 months, &gt, 12 months. AHIs were mostly caused by Staphylococcus aureus (39.2%) and streptococci (30.2%). EPIs were characterized by a preponderance of virulent microorganisms (S. aureus, Gram-negative bacilli (GNB), enterococci), MDROs (24%) and polymicrobial infections (27.4%). Conversely, coagulase-negative staphylococci (CoNS) and Cutibacterium species were predominant in LCIs (54.5% and 6.1%, respectively) and PICs (57.1% and 15.1%). The percentage of MDROs isolated in EPIs was more than three times the percentage isolated in LCIs (7.8%) and more than twice the proportion found in AHI (10.9%). There was a significant decreasing linear trend over the four time intervals post-surgery for virulent microorganisms, MDROs, and polymicrobial infections, and a rising trend for CoNS, streptococci and Cutibacterium spp. The observed differences have important implications for the empirical antimicrobial treatment of PJIs.

In this study, axenic cultures of sulfate-reducing (SRB) and nitrate-reducing (NRB) bacteria were examined for their ability to methylate inorganic tin and to methylate or dealkylate butyltin compounds. Environmentally relevant concentrations of natural abundance tributyltin (TBT) and 116Sn-enriched inorganic tin were added to bacterial cultures to identify bacterial-mediated methylation and dealkylation reactions. The results show that none of the Desulfovibrio strains tested was able to induce any transformation process. In contrast, Desulfobulbus propionicus strain DSM-6523 degraded TBT either under sulfidogenic or non-sulfidogenic conditions. In addition, it was able to alkykate 116Sn-enriched inorganic tin leading to the formation of more toxic dimethyltin and trimethyltin. A similar capacity was observed for incubations of Pseudomonas but with a much greater dealkykation of TBT. As such, Pseudomonas sp. ADR42 degraded 61% of the initial TBT under aerobic conditions and 35% under nitrate-reducing conditions. This is the first work reporting a simultaneous TBT degradation and a methylation of both inorganic tin species and TBT dealkykation products by SRB and NRB under anoxic conditions. These reactions are environmentally relevant as they can control the mobility of these compounds in aquatic ecosystems; as well as their toxicity toward resident organisms. © 2018 Elsevier P.R.G. would like to acknowledge the postdoctoral fellowship from the “Secretaria de Estado de Universidades e Investigacion” of the Spanish Ministry of Education and Science. P.N. would like to acknowledge the Doctoral fellowship from the Basque Country. This project was funded by French National Research Agency ( ANR – Jeune Chercheur program) in the framework of the MINAMETA project ( JCJC2006-0044 ). T. Stoichev is grateful to the Foundation for Science and Technology for fellowship SFRH/BPD/88675/2012 , co-financed by Programa Operacional Potencial Humano (POPH) /Fundo Social Europeu (FSE). Appendix A

Standard microorganism isolating technology applied for complex multiphase environmental samples such as soil or sediment needs pre-treatment steps to remove living cells from their mixed-phase microniche, by creating a liquid-phase sample. This process removes synergetic relationships, which help to maintain viability of yet-to-be-cultured and hard-to-culture bacteria. In this paper we demonstrate a high throughput Laser Micro-Sampling (LMS) technology for direct isolation of pure microbial cultures and microbial consortia from soil. This technology is based on laser printing of soil microparticles by focusing near-infrared laser pulses on specially prepared samples of a soil/gel mixture spread onto a gold-coated glass plate. Microsamples of soil are printed on glucose-peptone-yeast agar plates, to estimate the LMS process influence on functional and taxonomic microbial diversity, and on «Eco-log» sole carbon sources microplates, to investigate functional diversity by “metabolic fingerprinting”. The obtained results are compared with traditionally treated soil samples. It was shown that LMS treatment leads to increasing of cultured biodiversity and modifies the functional diversity. The strain of rare genus Nonomuraea was isolated by LMS from complex natural environment without using media selective for this genus. Graphical abstract

Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation within a species; however, few investigations demonstrate how naturally occurring SNPs may increase strain virulence. We recently used group A Streptococcus as a model pathogen to study bacteria strain genotype-patient disease phenotype relationships. Whole-genome sequencing of approximately 800 serotype M59 group A Streptococcus strains, recovered during an outbreak of severe invasive infections across North America, identified a disproportionate number of SNPs in the gene encoding multiple gene regulator of group A Streptococcus (mga). Herein, we report results of studies designed to test the hypothesis that the most commonly occurring SNP, encoding a replacement of arginine for histidine at codon 201 of Mga (H201R), significantly increases virulence. Whole transcriptome analysis revealed that the H201R replacement significantly increased expression of mga and 54 other genes, including many proven virulence factors. Compared to the wild-type strain, a H201R isogenic mutant strain caused significantly larger skin lesions in mice. Serial quantitative bacterial culture and noninvasive magnetic resonance imaging also demonstrated that the isogenic H201R strain was significantly more virulent in a nonhuman primate model of joint infection. These findings show that the H201R replacement in Mga increases the virulence of M59 group A Streptococcus and provide new insight to how a naturally occurring SNP in bacteria contributes to human disease phenotypes. Copyright © 2015 American Society for Investigative Pathology.