Search

Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to heat damage to cellular macromolecules. We here report that, in the presence of rapamycin, a potent inhibitor of Target of Rapamycin Complex 1 (TORC1), the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that usually arrest its growth. Consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance, suggesting that TORC1 restricts fission yeast growth at high temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens, which identified additional factors that suppress cell proliferation at high temperatures. Among them is Mks1, which is phosphorylated in a TORC1-dependent manner, forms a complex with the 14-3-3 protein Rad24, and suppresses the high-temperature growth independently of Sck1. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology.

Modern molecular microbiology elucidates the organizational principles of bacterial biofilms via detailed examination of the interplay between signaling and gene regulation. A complementary biophysical approach studies the mesoscopic dependencies at the cellular and multicellular levels with a distinct focus on intercellular forces and mechanical properties of whole biofilms. Here, motivated by recent advances in biofilm research and in other, seemingly unrelated fields of biology and physics, we propose a perspective that links the biofilm, a dynamic multicellular organism, with the physical processes occurring in the extracellular milieu. Using Bacillus subtilis as an illustrative model organism, we specifically demonstrate how such a rationale explains biofilm architecture, differentiation, communication, and stress responses such as desiccation tolerance, metabolism, and physiology across multiple scales--from matrix proteins and polysaccharides to macroscopic wrinkles and water-filled channels. [ABSTRACT FROM AUTHOR]

Sewage sludge in cities of Yangzi River Belt, China, generally exhibits a lower organic content and higher silt contentdue to leakage of drainage system, which caused low bioenergy recovery and carbon emission benefits in conventional anaerobic digestion (CAD). Therefore, this paper is on a pilot scale, a bio-thermophilic pretreatment anaerobic digestion (BTPAD) for low organic sludge (volatile solids (VS) of 4%) was operated with a long-term continuous flow of 200 days. The VS degradation rate and CH 4 yield of BTPAD increased by 19.93% and 53.33%, respectively, compared to those of CAD. The analysis of organic compositions in sludge revealed that BTPAD mainly improved the hydrolysis of proteins in sludge. Further analysis of microbial community proportions by high-throughput sequencing revealed that the short-term bio-thermophilic pretreatment was enriched in Clostridiales, Coprothermobacter and Gelria , was capable of hydrolyzing acidified proteins, and provided more volatile fatty acid (VFA) for the subsequent reaction. Biome combined with fluorescence quantitative polymerase chain reaction (PCR) analysis showed that the number of bacteria with high methanogenic capacity in BTPAD was much higher than that in CAD during the medium temperature digestion stage, indicating that short-term bio-thermophilic pretreatment could provide better methanogenic conditions for BTPAD. Furthermore, the greenhouse gas emission footprint analysis showed that short-term bio-thermophilic pretreatment could reduce the carbon emission of sludge anaerobic digestion system by 19.18%. [Display omitted] [ABSTRACT FROM AUTHOR]

The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic exchange predominantly using the kanR-rpsL cassette known as "Janus." The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. [ABSTRACT FROM AUTHOR]

Background Acinetobacter baumannii (Ab) disease in the United States is commonly attributed to outbreaks of 1 or 2 monophyletic carbapenem resistance (CR) Ab lineages that vary by region. However, there is limited knowledge regarding CR Ab epidemiology and population structures in the U.S. Deep South, and few studies compare contemporary CR and carbapenem-susceptible (Cs) Ab , despite relative prevalence of the latter. Methods We performed a multiyear analysis of 2462 Ab cases in a large healthcare system in Birmingham, AL, and 89 post-2021 Ab isolates were sequenced and phenotyped by antibiotic susceptibility tests. Results Although the cumulative CR rate was 17.7% in our cohort, rates regularly increased in winter months as result of seasonal changes in case incidence of Cs Ab , specifically. Genotyped CR Ab belonged to clonal group (CG) 1, CG2, CG108, CG250, or CG499, with local clones of CG108, CG250, and CG499 persisting over multiple months. There was no clonal expansion of any Cs Ab lineage. Among CR Ab isolates, levels of β-lactam antibiotic resistance and the repertoire of related genetic resistance determinants, which included the novel CR-conferring FtsI A515V polymorphism, differed according to CG. CG108 and CG499 isolates displayed specific heteroresistance to sulbactam and trimethoprim/sulfamethoxazole, respectively, which resulted in discrepant susceptibility results in microbroth versus agar-based antibiotic susceptibility tests modalities. Conclusions We report an unusually high degree of CR Ab phylogenetic diversity principally driven by emergent U.S. lineages harboring novel resistance elements that must be incorporated into diagnostic, surveillance, and preclinical research efforts. [ABSTRACT FROM AUTHOR]

Agriculture has recently prioritized Plant Growth Promoting Rhizobacteria (PGPR) due to their significance in the rhizosphere, which is an ecological unit in the biosphere. Isolating PGPR from Cicer arietinum (chickpea) nodules and characterizing its PGP activities was the purpose of the present study. The research showed that the specific strain could manufacture several useful chemicals, such as hydrogen cyanide (HCN), ammonia, siderophore, indole acetic acid (IAA) and nitrogenase. Furthermore, the isolate was identified as Bacillus licheniformis AS11 through 16s rRNA analysis, and alignment analysis showed 99% similarity with the Bacillus licheniformis KPA12 isolate. It's worth highlighting that, Bacillus licheniformis AS11 a potential PGPR, is considered a valuable asset for agriculture when it has a positive effect on plant growth. Therefore, Bacillus licheniformis AS11 can be a beneficial and constructive addition to the field of agriculture. These findings suggest potential applications in sustainable agriculture by improving crop yield through natural growth-promoting mechanisms and reducing reliance on chemical fertilizers. [ABSTRACT FROM AUTHOR]

Assays that utilize nucleic acid amplification are prone to contamination from a variety of sources, including both target organisms and amplicon. Implementing effective systems for contamination monitoring as well as laboratory best practices are essential to any quality management system for molecular microbiology testing. A comprehensive contamination monitoring toolbox will be introduced here that includes guidance for environment swabbing, positivity rate monitoring, use of process controls, and monitoring complaints. As prevention of contamination is preferable, best practices for laboratory design, workflow, and personnel training will also be discussed. Lastly, a suggested plan for managing a contamination event will be proposed. The laboratory director should carefully assess the specific risks associated with testing performed in the laboratory and create a written plan for contamination monitoring, prevention, and management of contamination events. [ABSTRACT FROM AUTHOR]

Bacteria have a remarkable ability to sense environmental stresses and to respond to these stressors by adapting their metabolism and physiology. In recent publications, investigators have suggested that multiple stresses that cause cell death share the mechanistic feature of stimulating the formation of reactive oxygen species (ROS). A central piece of evidence cited in these claims is the ability of exogenous antioxidant compounds to mitigate stress‐related cell death. The validity of attributing a positive effect of exogenous antioxidants to ROS‐mediated stress is challenged by an important study by Korshunov and Imlay in this issue of Molecular Microbiology. This study reports biochemical data that convincingly show that some commonly used antioxidants quench oxidants orders of magnitude too slowly to have a significant effect on the concentration of ROS in the cell. Under conditions where antioxidants minimize cell death, they also slow growth. Significantly, slowing cell growth by other means has the same restorative effect as adding an antioxidant. Based on the solid biochemical and genetic data, Korshunov and Imlay make the case for discarding the use of antioxidants to diagnose conditions that generate increased internal ROS production. [ABSTRACT FROM AUTHOR]

Summary: Gene knockout disrupts cellular homeostasis, altering gene expression, and phenotypes. We investigated whether cells return to their pre-knockout transcriptomic state through adaptive evolution experiments on hap4Δ and ade1Δ yeast strains. Analysis revealed that genes with higher expression levels and more physical interaction partners in wild-type strains were more likely to be restored, suggesting that genes of significant functional importance have increased resilience to genetic perturbations. However, as the experiment progressed, most initially restored genes became unrestored. Over 60% of differentially expressed genes in knockout strains remained unrestored in evolved strains. Evolved strains exhibited distinct transcriptomic states, diverging from the original strain over time. Ribosome biogenesis components exhibited systematic sequential changes during the evolution. Our findings suggest the knockout strain transcriptomes struggle to return to the original state even after 28 days of culture. Instead, compensatory mechanisms lead to distinct suboptimal states, highlighting the complex transcriptomic dynamics following genetic perturbations.

Summary: Crystal structure of the secretory chorismate mutase protein of Mycobacterium tuberculosis (MtbCM) reveals presence of a proline rich region on its surface that serve as a recognition site for protein-protein interaction. This study shows that MtbCM upregulates IL-10 which favors M. tuberculosis by affecting PKCε-MKP-1-p38 MAPK signaling. MtbCM translocates to the Golgi-network where it interacts with AKAP9 via its SH3-binding domain to inhibit AKAP9-PKCε interaction and reducing PKCε phosphorylation. In the absence of phosphorylated PKCε, IRAK3 fails to stabilize MKP-1 resulting in higher p38 MAPK activation and IL-10 production. M. smegmatis expressing MtbCM survived better in infected mice. Mutation in SH3-binding domain ablated MtbCM-AKAP9 interaction resulting in IL-10 production and decreased bacterial survival. This study highlights the importance of SH3-binding domain in host-pathogen interaction and a role of MtbCM in modulation of cytokine response and mycobacterial virulence in addition to its role in shikimate pathway.

Summary: Nitrosophilus labii HRV44T is a thermophilic chemolithoautotroph possessing clade II type nitrous oxide (N2O) reductase (NosZ) that has an outstanding activity in reducing N2O to dinitrogen gas. Here, we attempt to understand molecular responses of HRV44T to N2O. Time course transcriptome and proteomic mass spectrometry analyses under anaerobic conditions revealed that most of transcripts and peptides related to denitrification were constitutively detected, even in the absence of any nitrogen oxides as electron acceptors. Gene expressions involved in electron transport to NosZ were upregulated within 3 h in response to N2O, rather than upregulation of nos genes. Two genes encoding Crp/Fnr transcriptional regulators observed upstream of nap and nor gene clusters had significant negative correlations with nosZ expression. Statistical path analysis further inferred a significant causal relationship between the gene expression of nosZ and that of one Crp/Fnr regulators. Our findings contribute to understanding the transcriptional regulation in clade II type N2O-reducers.

Introduction: the need to have rapid, sensitive and specific laboratory techniques for the detection of pathogens that are difficult to diagnose led to the creation of the Molecular Biology Laboratory at the Provincial Center of Hygiene, Epidemiology and Microbiology of Villa Clara. Objective: to characterize the evolution and importance of Molecular Microbiology in the Laboratory of the Provincial Center of Hygiene, Epidemiology and Microbiology of Villa Clara from June 2018 to December 2023. Methods: cross-sectional descriptive study carried out at the Villa Clara Molecular Biology Laboratory in the period from June 2018 to December 2023 based on the total samples processed by polymerase chain reaction, several technological platforms and various kits were used. commercial. Results: In 2018, molecular diagnosis was implemented with two platforms, Rotor Gene-Q® and Gene Xpert® MTB/RIF, and in 2021, four were available, when the SLAN and the COBAS 4800® platform were introduced. The laboratory became relevant due to the arrival of the 2019 new coronavirus pandemic: 511,225 nasopharyngeal exudate samples and 14,568 determinations were processed for the diagnosis of human immunodeficiency viruses, hepatitis C and B, and human papilloma carried out on the COBAS 4800® platform. In 2023, the pathogen detection capacity was strengthened and the community cocirculation of influenza A and B, parainfluenza virus type 4, the OC 43 coronavirus, SARS-CoV-2 and dengue was identified. Conclusions: the progressive increase in the resolution capacity of the Molecular Biology Laboratory, together with the implementation of new diagnostic platforms, has allowed us to strengthen the detection of pathogens of clinical interest and improve virological sentinel surveillance. [ABSTRACT FROM AUTHOR]

The supply of coconut crabs (Birgus latro Linnaeus, 1767) has been sourced from capture in the wild. Development efforts in aquaculture have succeeded in producing eggs. However, further development with larval maintenance failed because it was constrained by a low survival rate. This study aimed to identify the potential probiotic bacteria in coconut crabs. Coconut crab samples were collected from Moor Island, South Patani District, Central Halmahera Regency, North Maluku Province, Indonesia. The intestines of coconut crabs were taken as much as 1 g, then ground on a porcelain dish and added to Tri-Salt. The scouring liquid was taken in 0.1 ml and spread on a petri dish containing Nutrient Agar. The isolated used were marked with BL1, BL2, BL3, BL4, and BL5. Molecular identification was performed with sequencing and then analyzed using the Basic Local Alignment Search Tool (BLAST). The selection of isolates from the gastrointestinal tract of coconut crabs showed that the bacteria obtained were Enterobacter tabaci (BL1), Enterobacter hormaechei (BL2), Bacillus horneckiae (BL3), Pseudomonas stutzeri (BL4), and Acinetobacter variabilis (BL5). Further testing revealed that the bacteria A. variabilis (BL5) is a probiotic isolate in coconut crabs. [ABSTRACT FROM AUTHOR]

Summary: Stenotrophomonas maltophilia (Sm), a multidrug-resistant pathogen often isolated from immunocompromised individuals, presents its flagellin to multimeric tandem repeats within the ectodomain of mucin-1 (MUC1-ED), expressed on airway epithelia. Flagellated Sm increases neuraminidase-1 (NEU1) sialidase association with and desialylation of MUC1-ED. This NEU1-mediated MUC1-ED desialylation unmasks cryptic binding sites for Sm flagellin, increasing flagellin and Sm binding to airway epithelia. MUC1 overexpression increases receptor number whereas NEU1 overexpression elevates receptor binding affinity. Silencing of either MUC1 or NEU1 reduces the flagellin-MUC1 interaction. Sm/flagellin provokes MUC1-ED autoproteolysis at a juxtamembranous glycine-serine peptide bond. MUC1-ED shedding from the epithelium not only occurs in vitro, but in the bronchoalveolar compartments of Sm/flagellin-challenged mice and patients with ventilator-associated Sm pneumonia. Finally, the soluble flagellin-targeting, MUC1-ED decoy receptor dose-dependently inhibits multiple Sm flagellin-driven pathogenic processes, in vitro, including motility, biofilm formation, adhesion, and proinflammatory cytokine production, and protects against lethal Sm lung infection, in vivo.

Summary: Halophiles, thriving in harsh saline environments, capture scientific interest due to their remarkable ability to prosper under extreme salinity. This study unveils the distinct salt-induced activation of methionine sulfoxide reductases (MsrA) from Halobacterium hubeiense, showcasing a significant enhancement in enzymatic activity across various salt concentrations ranging from 0.5 to 3.5 M. This contrasts sharply with the activity profiles of non-halophilic counterparts. Through comprehensive molecular dynamics simulations, we demonstrate that salt ions stabilize and compact the enzyme’s structure, notably enhancing its substrate affinity. Mutagenesis analysis further confirms the essential role of salt bridges formed by the basic Arg168 residue in salt-induced activation. Mutating Arg168 to an acidic or neutral residue disrupts salt-induced activation, substantially reducing the enzyme activity under salt conditions. Our research provides evidence of salt-activated MsrA activity in halophiles, elucidating the molecular basis of halophilic enzyme activity in response to salts.

Summary: 4-Methylcytosine (4mC) and 6-methyladenine (6mA) are the most prevalent types of DNA modifications in prokaryotes. However, whether there is crosstalk between 4mC and 6mA remain unknown. Here, methylomes and transcriptomes of Geobacter sulfurreducens exposed to different intensities of extremely low frequency electromagnetic fields (ELF-EMF) were investigated. Results showed that the second adenine of all the 5′-GTACAG-3′ motif was modified to 6mA (M-6mA). For the other 6mA (O-6mA), the variation in their distance from the neighboring M-6mA increased with the intensity of ELF-EMF. Moreover, cytosine adjacent to O-6mA has a much higher probability of being modified to 4mC than cytosine adjacent to M-6mA, and the closer an unmodified cytosine is to 4mC, the higher the probability that the cytosine will be modified to 4mC. Furthermore, there was no significant correlation between DNA methylation and gene expression regulation. These results suggest a reference signal that goes from M-6mA to O-6mA to 4mC.

Summary: The mechanistic target of rapamycin (mTOR) positively regulates multiple steps of the HIV-1 replication cycle. We previously reported that a 12-week supplementation of antiretroviral therapy (ART) with metformin, an indirect mTOR inhibitor used in type-2 diabetes treatment, reduced mTOR activation and HIV transcription in colon-infiltrating CD4+ T cells, together with systemic inflammation in nondiabetic people with HIV-1 (PWH). Herein, we investigated the antiviral mechanisms of metformin. In a viral outgrowth assay performed with CD4+ T cells from ART-treated PWH, and upon infection in vitro with replication-competent and VSV-G-pseudotyped HIV-1, metformin decreased virion release, but increased the frequency of productively infected CD4lowHIV-p24+ T cells. These observations coincided with increased BST2/tetherin (HIV release inhibitor) and Bcl-2 (pro-survival factor) expression, and improved recognition of productively infected T cells by HIV-1 envelope antibodies. Thus, metformin exerts pleiotropic effects on post-integration steps of the HIV-1 replication cycle and may be used to accelerate viral reservoir decay in ART-treated PWH.

Summary: The interplay between lipid metabolism and immune response in macrophages plays a pivotal role in various infectious diseases, notably tuberculosis (TB). Herein, we illuminate the modulatory effect of heat-killed Mycobacterium tuberculosis (HKMT) on macrophage lipid metabolism and its implications on the inflammatory cascade. Our findings demonstrate that HKMT potently activates the lipid scavenger receptor, CD36, instigating lipid accumulation. While CD36 inhibition mitigated lipid increase, it unexpectedly exacerbated the inflammatory response. Intriguingly, this paradoxical effect was linked to an upregulation of PPARδ. Functional analyses employing PPARδ modulation revealed its central role in regulating both lipid dynamics and inflammation, suggesting it as a potential therapeutic target. Moreover, primary monocytic cells from diabetic individuals, a demographic at amplified risk of TB, exhibited heightened PPARδ expression and inflammation, further underscoring its pathological relevance. Targeting PPARδ in these cells effectively dampened the inflammatory response, offering a promising therapeutic avenue against TB.

This article, titled "Editorial: Systemic inflammation in severe infectious diseases," explores the topic of systemic inflammation in the context of severe infectious diseases. The research compiled in this article provides an in-depth examination of the mechanisms, biomarkers, and therapeutic strategies aimed at understanding and mitigating the detrimental effects of systemic inflammation. The article discusses the role of biomarkers in predicting disease severity and guiding therapeutic interventions, as well as innovative approaches to modulate the inflammatory response. It also highlights emerging concepts and future directions in the field. Overall, this article contributes to our understanding of systemic inflammation and its management in severe infectious diseases. [Extracted from the article]

The 2024 National Convention hosted by the Mu Omicron Chapter of Columbus State University took place in Columbus, Georgia. The event included presentations, field trips, an awards banquet, and a business meeting where new Executive Committee positions were announced. Various awards were given out for oral and poster presentations, and the convention concluded with positive feedback from attendees. The next convention in 2026 will be held at Florida State University in Tallahassee, Florida, hosted by the Tau Sigma chapter. [Extracted from the article]

Understanding that evolution progresses through generation of DNA variants followed by selection is a key learning outcome for biology students. We designed an integrated and innovative undergraduate laboratory exercise using Saccharomyces cerevisiae to demonstrate these principles. Students perform in vitro experimental evolution by repeatedly propagating large or small yeast colonies on a weekly basis. Small-colony variants known as petites arise by mutations that disrupt aerobic respiration. To demonstrate the effects of increased mutation rates, half of the selection lines are exposed to ultraviolet irradiation. To understand how the petite phenotype arises, polymerase chain reaction (PCR) is performed to examine mitochondrial DNA, while biochemical assays are used to assess the ability of petites to undergo aerobic respiration. This exercise demonstrates evolution by artificial selection over a suitably short timeframe and links the results to a critical biochemical process: the role of mitochondria in aerobic respiration and ATP production. By implementing these experiments, we successfully demonstrated that the frequencies of petite mutants in evolved populations varied according to the selection pressure we applied, and that petite mutants carried deletions in mitochondrial DNA as anticipated. Through an integrated learning context, this practical exercise promotes fundamental understanding of evolutionary processes and fosters critical thinking skills. [ABSTRACT FROM AUTHOR]

Paratuberculosis (Johne's disease) is a chronic granulomatous small intestine disease caused by MAP. Diagnosing and isolating infected animals is the most important measure for controlling the disease. Therefore, this study aimed to molecularly identify mycobacterium isolated from ELISA- positive cows with Johne's disease by nested PCR from the samples from Markazi Province, Iran. For this purpose, 2938 samples were decontaminated and then cultured on the Herrold egg culture medium containing mycobactin and no mycobactin. After DNA extraction, PCR for 16S rRNA was first performed, followed by nested PCR on positive samples. Of 2938 samples, 87 were positive, and 26 were suspected. All positive isolates were observed in Ziehl-Neelsen staining in microscopic expansion. A 543-bp band was observed in 26 tested samples and mycobacterium strains in PCR for 16S rRNA, indicating the presence of mycobacterium in the above samples. Nested PCR was performed for all isolates and positive and negative control strains. A 398-bp band was obtained in the first stage, and a 298-bp fragment was obtained in the second stage, indicating the presence of MAP in the samples. Accordingly, nested PCR is suggested as a proper method for the quick and definitive diagnosis of disease cases. [ABSTRACT FROM AUTHOR]

Plasmids are commonly used tools in microbiology and molecular biology and have important and wide-ranging applications in the study of gene function, protein expression, and compound synthesis. The complex relationship between necessary antibiotic addition, compatibility between multiple plasmids, and the growth burden of host bacteria has plagued the wider use of compatibility plasmids. In this study, we constructed the terminal polymerization pathway of PSA by exogenously expressing the neuA, neuD, and neuS genes after the knockdown of Eschesrichia coli BL21 (DE3). Duet series vectors were utilized to regulate the expression level of neuA, neuD, and neuS genes to study the gene expression level, plasmid copy number growth burden, pressure of antibiotic addition, stability of compatible plasmids, and the level of expression stability of exogenous genes, as well as the effect on the biological reaction process. The results showed that the three genes, neuA, neuD, and neuS, were enhanced in the recombinant strain E. coli NA-05, with low copy, medium copy, and high copy, respectively. The effect of PSA synthesis under standard antibiotic pressure was remarkable. The results of this thesis suggest the use of a Duet series of compatible expression vectors to achieve the stable existence and co-expression of multiple genes in recombinant bacteria, which is a good reason for further research. [ABSTRACT FROM AUTHOR]

Summary: Microorganisms are critical to the stability of aquatic environments, and understanding the ecological mechanisms of microbial community is essential. However, the distinctions and linkages across biogeographic patterns, ecological processes, and formation mechanisms of microbes in rivers and lakes remain unknown. Accordingly, microbiome-centric analysis was conducted in rivers and lakes in the Yangtze River watershed. Results revealed significant differences in the structure and diversity of microbial communities between rivers and lakes, with rivers showing higher diversity. Lakes exhibited lower community stability, despite higher species interactions. Although deterministic processes dominated microbial community assembly both in rivers and lakes, higher stochastic processes of rare and abundant taxa exhibited in rivers. Spatial factors influenced river microbial community, while environmental factors drove differences in the lake bacterial community. This study deepened the understanding of microbial biogeography and formation mechanisms in large watershed rivers and lakes, highlighting distinct community aggregation patterns between river and lake microorganisms.

Summary: Type I interferon (IFN) production is crucial in tuberculosis pathogenesis, yet the bacterial factors initiating this process are incompletely understood. CpsA, protein of Mycobacterium marinum and Mycobacterium tuberculosis, plays a key role in maintaining bacterial virulence and inhibiting host cell LC3-associated phagocytosis. By utilizing CpsA full deletion mutant studies, we re-verified its essential role in infection-induced pathology and revealed its new role in type I IFN expression. CpsA deficiency hindered IFN production in infected macrophages in vitro as well as zebrafish and mice in vivo. This effect was linked to the cGAS-TBK1-IRF3 pathway, as evidenced by decreased TBK1 and IRF3 phosphorylation in CpsA-deficient bacterial strain-infected macrophages. Moreover, we further show that CpsA deficiency cause decreased cytosolic DNA levels, correlating with impaired phagosomal membrane rupture. Our findings reveal a new function of mycobacterial CpsA in type I IFN production and offer insight into the molecular mechanisms underlying mycobacterial infection pathology.

Summary: Programmed cell death (PCD) is tightly orchestrated by molecularly defined executors and signaling pathways. NLRP6, a member of cytoplasmic pattern recognition receptors, has a multifaceted role in host resistance to bacterial infection. However, whether and how NLRP6 may contribute to regulate host PCD during Gram-negative bacterial infection remain to be illuminated. Here, we report that NLRP6 promotes RIP1 kinase-mediated necroptosis, a form of lytic PCD, in both an in vitro and in vivo model of Salmonella typhimurium infection. By downregulating TAK1-mediated p38MAPK/MK2 phosphorylation, NLRP6 decreased RIP1 phosphorylation at residue S321 and subsequently increased RIP1 kinase-dependent MLKL phosphorylation. Suppression of p38MAPK/MK2 cascade not only reduced the number of dead cells caused by NLRP6 but also decreased the systemic dissemination of S. typhimurium resulting from NLRP6. Taken together, our findings provide new insights into the role and regulatory mechanism of NLRP6-associated antimicrobial responses by revealing a function for NLRP6 in regulating necroptosis.

Abstract Background No univocal recommendation exists for microbiological diagnosis of ventilator-associated pneumonia (VAP). Sampling of either proximal or distal respiratory tract likely impacts on the broad range of VAP incidence between cohorts. Immune biomarkers to rule-in/rule-out VAP diagnosis, although promising, have not yet been validated. COVID-19-induced ARDS made VAP recognition even more challenging, often leading to overdiagnosis and overtreatment. We evaluated the impact of different respiratory samples and laboratory techniques on VAP incidence and microbiological findings in COVID-19 patients. Methods Prospective single-centre cohort study conducted among COVID-19 mechanically ventilated patients in Policlinico Hospital (Milan, Italy) from January 2021 to May 2022. Microbiological confirmation of suspected VAP (sVAP) was based on concomitant endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL). Conventional and fast microbiology (FILMARRAY® Pneumonia Panel plus, BALFAPPP) as well as immunological markers (immune cells and inflammatory cytokines) was analysed. Results Seventy-nine patients were included. Exposure to antibiotics and steroid therapy before ICU admission occurred in 51/79 (64.6%) and 60/79 (65.9%) patients, respectively. Median duration of MV at VAP suspicion was 6 (5–9) days. Incidence rate of microbiologically confirmed VAP was 33.1 (95% CI 22.1–44.0) and 20.1 (95% CI 12.5–27.7) according to ETA and BAL, respectively. Concordance between ETA and BAL was observed in 35/49 (71.4%) cases, concordance between BALFAPPP and BAL in 39/49 (79.6%) cases. With BAL as reference standard, ETA showed 88.9% (95% CI 70.8–97.7) sensitivity and 50.0% (95% CI 28.2–71.8) specificity (Cohen’s Kappa 0.40, 95% CI 0.16–0.65). BALFAPPP showed 95.0% (95% CI 75.1–99.9) sensitivity and 69% (95% CI 49.2–84.7) specificity (Cohen’s Kappa 0.60, 95% CI 0.39–0.81). BAL IL-1β differed significantly between VAP (135 (IQR 11–450) pg/ml) and no-VAP (10 (IQR 2.9–105) pg/ml) patients (P = 0.03). Conclusions In COVID-19 ICU patients, differences in microbial sampling at VAP suspicion could lead to high variability in VAP incidence and microbiological findings. Concordance between ETA and BAL was mainly limited by over 20% of ETA positive and BAL negative samples, while BALFAPPP showed high sensitivity but limited specificity when evaluating in-panel targets only. These factors should be considered when comparing results of cohorts with different sampling. BAL IL-1β showed potential in discriminating microbiologically confirmed VAP. Clinical Trial registration: NCT04766983, registered on February 23, 2021.

Summary: Streptococcus suis is a zoonotic pathogen that can cause meningitis and septicaemia. The consumption of undercooked pig products is an important risk factor for zoonotic infections, suggesting an oral route of infection. In a human enteroid model, we show that the zoonotic CC1 genotype has a 40% higher translocation frequency than the non-zoonotic CC16 genotype. Translocation occurred without increasing the permeability or disrupting the adherens junctions and tight junctions of the epithelial monolayer. The translocation of zoonotic S. suis was correlated with the presence of Gb3-positive cells, a human glycolipid receptor found on Paneth cells and targeted by multiple enteric pathogens. The virulence factors Streptococcal adhesin Protein and suilysin, known to interact with Gb3, were not essential for translocation in our epithelial model. Thus, the ability to translocate across an enteroid monolayer correlates with S. suis core genome composition and the presence of Gb3-positive cells in the intestinal epithelium.

Summary: Cells cease to proliferate above their growth-permissible temperatures, a ubiquitous phenomenon generally attributed to heat damage to cellular macromolecules. We here report that, in the presence of rapamycin, a potent inhibitor of Target of Rapamycin Complex 1 (TORC1), the fission yeast Schizosaccharomyces pombe can proliferate at high temperatures that usually arrest its growth. Consistently, mutations to the TORC1 subunit RAPTOR/Mip1 and the TORC1 substrate Sck1 significantly improve cellular heat resistance, suggesting that TORC1 restricts fission yeast growth at high temperatures. Aiming for a more comprehensive understanding of the negative regulation of high-temperature growth, we conducted genome-wide screens, which identified additional factors that suppress cell proliferation at high temperatures. Among them is Mks1, which is phosphorylated in a TORC1-dependent manner, forms a complex with the 14-3-3 protein Rad24, and suppresses the high-temperature growth independently of Sck1. Our study has uncovered unexpected mechanisms of growth restraint even below the temperatures deleterious to cell physiology.

Summary: Host range of a phage is determined at the various life cycle stages during phage infection. We reported the limited phage-receptor interaction between the RNA phage, PP7 and its host Pseudomonas aeruginosa strains: PAO1 has susceptible type IV pilus (TFP) pilin, whereas PA14 has resistant pilin. Here, we have created a PA14 derivative (PA14P) with the PAO1 pilin gene and found that other determinants than TFP pilin could limit PP7 infectivity in PA14P. Transposon mutant screens revealed that PP7 infectivity was restored in the PA14P mutants (htrB2) lacking a secondary acyltransferase in lipid A biosynthesis. The lack of this enzyme increased the RNA phage entry, which is deemed attributed to the loosened lipopolysaccharide (LPS) structure. Polymyxin B treatment also selectively increased the RNA phage entry. These results demonstrated that LPS structures could limit the entry stage of RNA phages, providing another determinant for the host range in diverse P. aeruginosa strains.

Hepatitis C virus (HCV) can cause both acute and chronic hepatitis infections. Gaziantep is located southeast part of Turkey and has a border with Syria. More than 400,000 Syrian refugees live in Gaziantep. The aim of this study was to evaluate distribution of HCV genotypes among Syrian patients and in people who inject drugs. Serum samples form 1,628 individuals (786 female, 842 male) which were sent to our laboratory for genotyping between January 2013 and December 2022, were analyzed retrospectively. Three different HCV genotyping assays (Qiagen, RTA and Abbott) were used during the 10-year study period. Out of the 1,628 patients, genotype 1 was detected in 51.5%, genotype 3 in 21.4%, genotype 4 in 20%, genotype 5 in 4.6%, genotype 2 in 1.3%. Mixed genotype was found in 20 patients. Of the patients, 1,143 were Turkish patients and among those patients genotype 1 (66.8%) was the most common genotype followed by genotype 3 (29%). Among Syrian patients (n = 477), genotype 4 (64.2%) was predominant genotype followed by genotype 1 and genotype 5. Genotype 3 was detected in 277 (79.6%) prisoners. All of them were male and probably the main source of HCV infection was intravenous drug abuse. While genotypes 1 and 4 were common in females, genotypes 1 and 3 were common in males. In the future genotype 3 may become an increasing problem due to the persons who inject drugs. Less frequent genotypes such as 4 and 5 may become more frequent due to Syrian patients. [ABSTRACT FROM AUTHOR]

A 66-year-old male patient diagnosed with small-cell lung cancer received chemotherapy and immunotherapy, resulting in successful tumor control. However, the patient subsequently experienced a fever and rapid progression of the pulmonary cavity. Despite sampling bronchoscopic bronchoalveolar lavage fluid for targeted next-generation sequencing (tNGS), the cause remained unidentified. Adding bronchoalveolar lavage fluid to sense metagenomic next-generation sequencing (mNGS) confirmed the infection caused by actinomycetes. The patient's condition improved after active anti-infection treatment. This case was further analyzed and discussed through a comprehensive literature review, focusing on molecular microbiological diagnosis and treatment processes. The points outlined were as follows: the advancement of molecular microbiology has gradually reduced the challenges associated with diagnosing rare infectious diseases such as pulmonary actinomycosis. Additionally, in immunodeficient individuals, certain infectious diseases with a chronic course may exhibit acute and aggressive characteristics, which is of concern to all colleagues. Currently, tNGS and mNGS are widely employed in clinical settings as practical tools for diagnosing infectious diseases. Notably, these two methods are not substitutes for each other but complement each other. [ABSTRACT FROM AUTHOR]

Advancements in DNA sequencing technologies within the last decade have stimulated an unprecedented interest in the human microbiome, largely due the broad diversity of human diseases found to correlate with microbiome dysbiosis. As a direct consequence of these studies, a vast number of understudied and uncharacterized microbes have been identified as potential drivers of mucosal health and disease. The looming challenge in the field is to transition these observations into defined molecular mechanistic studies of symbiosis and dysbiosis. In order to meet this challenge, many of these newly identified microbes will need to be adapted for use in experimental models. Consequently, this review presents a comprehensive overview of the molecular microbiology tools and techniques that have played crucial roles in genetic studies of the bacteria found within the human oral microbiota. Here, we will use specific examples from the oral microbiome literature to illustrate the biology supporting these techniques, why they are needed in the field, and how such technologies have been implemented. It is hoped that this information can serve as a useful reference guide to help catalyze molecular microbiology studies of the many new understudied and uncharacterized species identified at different mucosal sites in the body. [ABSTRACT FROM AUTHOR]

Introduction: The emergence of resistance and interference mechanisms to phage infection can hinder the success of bacteriophage-based applications, but the significance of these mechanisms in phage therapy has not been determined. This work studies the emergence of Salmonella isolates with reduced susceptibility to a cocktail of three phages under three scenarios: i) Salmonella cultures (LAB), ii) biocontrol of cooked ham slices as a model of food safety (FOOD), and iii) oral phage therapy in broilers (PT). Methods: S. Typhimurium ATCC 14028 RifR variants with reduced phage susceptibility were isolated from the three scenarios and conventional and molecular microbiology techniques were applied to study them. Results and discussion: In LAB, 92% of Salmonella isolates lost susceptibility to all three phages 24 h after phage infection. This percentage was lower in FOOD, with 4.3% of isolates not susceptible to at least two of the three phages after seven days at 4°C following phage treatment. In PT, 9.7% and 3.3 % of isolates from untreated and treated broilers, respectively, displayed some mechanism of interference with the life cycle of some of the phages. In LAB and FOOD scenarios, resistant variants carrying mutations in rfc and rfaJ genes involved in lipopolysaccharide synthesis (phage receptor) were identified. However, in PT, the significant decrease of EOP, ECOI, and burst size observed in isolates was prompted by lateral gene transfer of large IncI1 plasmids, which may encode phage defense mechanisms. These data indicate that the acquisition of specific conjugative plasmids has a stronger impact than mutagenesis on the emergence of reduced phage-susceptibility bacteria in certain environments. In spite of this, neither mechanism seems to significantly impair the success of Salmonella biocontrol and oral phage therapy. [ABSTRACT FROM AUTHOR]

In this mini-review, some of the most significant, from the authors' point of view, features of the methodology of Fourier transform infrared (FTIR) spectroscopy and interpretation of experimentally obtained spectroscopic data are considered, as applied to the molecular spectrochemical analysis of microbiological objects using examples published in the specialised scientific literature mainly in recent years. Examples of typical inaccuracies and errors both of a methodological nature and arising in the interpretation of spectroscopic data are also presented and briefly discussed. [ABSTRACT FROM AUTHOR]

Drought poses a significant threat to agricultural food production, particularly in the cultivation of rice, which is semiaquatic plant. Developing drought resistant rice varieties through conventional breeding approaches is a challenging task due to the complex genetics underlying drought resistance. The mechanisms underlying drought resistance in plants are intricate and encompass a range of physiological and biochemical phenomena, contributing to the complexity of the breeding process. The inherent complexity of drought resistance necessitates the intervention of molecular mechanisms in breeding programs. This review aims to shed light on the current molecular breeding methods employed in the development of durable drought resistant rice varieties. [ABSTRACT FROM AUTHOR]

The article in Molecular Microbiology explores neglected tropical diseases (NTDs) caused by various pathogens in tropical regions, impacting around one billion people in Africa, Asia, and Latin America. Understanding host-pathogen interactions is crucial for developing new treatments for these diseases. The article highlights research on the molecular mechanisms of pathogen-host interactions and the strategies pathogens use to infect hosts, evade immune responses, and spread infections. Studies in the special issue focus on the molecular biology of NTDs to improve diagnosis and treatment, offering hope for new solutions to combat these diseases. [Extracted from the article]

This article, published in the journal Molecular Microbiology, focuses on the mechanisms of host cell exit by intracellular pathogens. While much is known about how pathogens enter host cells, less is understood about how they exit. The article highlights a collection of research articles, resource articles, and reviews that explore the exit mechanisms of bacterial, fungal, and parasitic pathogens. The authors discuss the three main pathways of host cell exit: membrane-dependent exit without host cell lysis, active lytic destruction of the host cell, and the initiation of programmed cell death. The article also mentions the funding and collaborative efforts of the research network SPP 2225, which aims to study exit strategies of intracellular pathogens. Overall, this collection of articles provides valuable insights into the exit mechanisms of various pathogens and may stimulate further research in this field. [Extracted from the article]

We aimed to systematize and assess scientific information on tetracycline (TET) resistance genes in animals, products, and by-products in the Latin America and the Caribbean (LAC) region. PRISMA guidelines were followed. Only original articles published in peer-reviewed journals were considered. Sixty articles published between 2003 and 2023 met the inclusion criteria. The geographical areas of study were Brazil, Mexico, Chile, and Costa Rica, and, to a lesser extent, Colombia, Bolivia, Cuba, Jamaica, Puerto Rico, and Uruguay. The studies were related to livestock, wild animals, and pets. The most common isolated bacteria were Escherichia coli and Salmonella spp. The tet genes found in higher frequency in the samples or isolates evaluated were tetA, tetB, tetM, tetL, tetK, tetC, tetO, tetD, tetG, tetW, tetS, tetQ, tetE, tetH, tetJ, tetZ, and tetY. Studies evaluating the presence of tet genes in animals in LAC are limited despite TET being antibiotics widely used in animals. It is necessary to establish cross border public policies that allow the constant training of medical and related personnel regarding the responsible use of antibiotics in animals and the effective monitoring of the phenomenon in the region.

Summary: Reactive oxygen species (ROS) produced as a secondary effect of bactericidal antibiotics are hypothesized to play a role in killing bacteria. If correct, ROS may play a role in development of de novo resistance. Here we report that single-gene knockout strains with reduced ROS scavenging exhibited enhanced ROS accumulation and more rapid acquisition of resistance when exposed to sublethal levels of bactericidal antibiotics. Consistent with this observation, the ROS scavenger thiourea in the medium decelerated resistance development. Thiourea downregulated the transcriptional level of error-prone DNA polymerase and DNA glycosylase MutM, which counters the incorporation and accumulation of 8-hydroxy-2′-deoxyguanosine (8-HOdG) in the genome. The level of 8-HOdG significantly increased following incubation with bactericidal antibiotics but decreased after treatment with the ROS scavenger thiourea. These observations suggest that in E. coli sublethal levels of ROS stimulate de novo development of resistance, providing a mechanistic basis for hormetic responses induced by antibiotics.

ABSTRACTIntestinal dysbiosis can lead to the induction of systemic immune-mediated inflammatory diseases, such as Crohn’s disease Although archaea are part of the commensal microbiota, they are still one of the least studied microorganisms. The aim of our study was the standardization of the optimal conditions and primers for sequencing of the gut archaeome using Next Generation Sequencing, and evaluation of the differences between the composition of archaea in patients and healthy volunteers, as well as analysis of the changes that occur in the archaeome of patients depending on disease activity. Newly diagnosed patients were characterized by similar archeal profiles at every taxonomic level as in healthy individuals (the dominance of Methanobacteria at the class level, and Methanobrevibacter at the genus level). In turn, in patients previously diagnosed with Crohn’s disease (both in active and remission phase), an increased prevalence of Thermoplasmata, Thermoprotei, Halobacteria (at the class level), and Halococcus, Methanospaera or Picrophilus (at the genus level) were observed. Furthermore, we have found a significant correlation between the patient’s parameters and the individual class or species of Archaea. Our study confirms changes in archaeal composition in pediatric patients with Crohn’s disease, however, only in long-standing disease. At the beginning of the disease, the archeal profile is similar to that of healthy people. However, in the chronic form of the disease, significant differences in the composition of archaeome begin to appear. It seems that some archaea may be a good indicator of the chronicity and activity of Crohn’s disease.

Background: No univocal recommendation exists for microbiological diagnosis of ventilator-associated pneumonia (VAP). Sampling of either proximal or distal respiratory tract likely impacts on the broad range of VAP incidence between cohorts. Immune biomarkers to rule-in/rule-out VAP diagnosis, although promising, have not yet been validated. COVID-19-induced ARDS made VAP recognition even more challenging, often leading to overdiagnosis and overtreatment. We evaluated the impact of different respiratory samples and laboratory techniques on VAP incidence and microbiological findings in COVID-19 patients. Methods: Prospective single-centre cohort study conducted among COVID-19 mechanically ventilated patients in Policlinico Hospital (Milan, Italy) from January 2021 to May 2022. Microbiological confirmation of suspected VAP (sVAP) was based on concomitant endotracheal aspirates (ETA) and bronchoalveolar lavage (BAL). Conventional and fast microbiology (FILMARRAY® Pneumonia Panel plus, BALFAPPP) as well as immunological markers (immune cells and inflammatory cytokines) was analysed. Results: Seventy-nine patients were included. Exposure to antibiotics and steroid therapy before ICU admission occurred in 51/79 (64.6%) and 60/79 (65.9%) patients, respectively. Median duration of MV at VAP suspicion was 6 (5–9) days. Incidence rate of microbiologically confirmed VAP was 33.1 (95% CI 22.1–44.0) and 20.1 (95% CI 12.5–27.7) according to ETA and BAL, respectively. Concordance between ETA and BAL was observed in 35/49 (71.4%) cases, concordance between BALFAPPP and BAL in 39/49 (79.6%) cases. With BAL as reference standard, ETA showed 88.9% (95% CI 70.8–97.7) sensitivity and 50.0% (95% CI 28.2–71.8) specificity (Cohen's Kappa 0.40, 95% CI 0.16–0.65). BALFAPPP showed 95.0% (95% CI 75.1–99.9) sensitivity and 69% (95% CI 49.2–84.7) specificity (Cohen's Kappa 0.60, 95% CI 0.39–0.81). BAL IL-1β differed significantly between VAP (135 (IQR 11–450) pg/ml) and no-VAP (10 (IQR 2.9–105) pg/ml) patients (P = 0.03). Conclusions: In COVID-19 ICU patients, differences in microbial sampling at VAP suspicion could lead to high variability in VAP incidence and microbiological findings. Concordance between ETA and BAL was mainly limited by over 20% of ETA positive and BAL negative samples, while BALFAPPP showed high sensitivity but limited specificity when evaluating in-panel targets only. These factors should be considered when comparing results of cohorts with different sampling. BAL IL-1β showed potential in discriminating microbiologically confirmed VAP. Clinical Trial registration: NCT04766983, registered on February 23, 2021. [ABSTRACT FROM AUTHOR]

Tuberculous meningitis (TBM) is the most common type of central nervous system tuberculosis (TB) and has the highest mortality and disability rate. Early diagnosis is key to improving the prognosis and survival rate of patients. However, laboratory diagnosis of TBM is often difficult due to its paucibacillary nature and sub optimal sensitivity of conventional microbiology and molecular tools which often fails to detect the pathogen. The gold standard for TBM diagnosis is the presence of MTB in the CSF. The recognised methods for the identification of MTB are acidfast bacilli (AFB) detected under CSF smear microscopy, MTB cultured in CSF, and MTB detected by polymerase chain reaction (PCR). Currently, many studies consider that all diagnostic techniques for TBM are not perfect, and no single technique is considered simple, fast, cheap, and efficient. A definite diagnosis of TBM is still difficult in current clinical practice. In this review, we summarise the current state of microbiological and molecular biological diagnostics for TBM, the latest advances in research, and discuss the advantages of these techniques, as well as the issues and challenges faced in terms of diagnostic effectiveness, laboratory infrastructure, testing costs, and clinical expertise, for clinicians to select appropriate testing methods. [ABSTRACT FROM AUTHOR]

• Largest systematic comparison of posted versus fresh sputum microbiology in CF exploring the impact on both conventional culture as well as molecular identification. • Posted sputum samples reliably reproduced culture-based and molecular microbiology of freshly collected samples, even after prolonged delays at ambient conditions. • Study provides reassurance for samples posted from home can be relied upon for clinical interpretation. Regular surveillance microbiology of sputum is used in cystic fibrosis (CF) to monitor for new pathogens and target treatments. A move to remote clinics has meant greater reliance on samples collected at home and posted back. The impact of delays and sample disruption caused by posting has not been systematically assessed but could have significant implications for CF microbiology. Sputum samples collected from adult CF patients were mixed, split, and either processed immediately or posted back to laboratory. Processing involved a further split into aliquots for culture-dependant and-independent microbiology (quantitative PCR [QPCR] and microbiota sequencing). We calculated retrieval by both approaches for five typical CF pathogens: Pseudomonas aeruginosa, Burkholderia cepacia complex, Achromobacter xylosoxidans, Staphylococcus aureus and Stenotrophomonas maltophilia. 93 paired samples were collected from 73 CF patients. Median interval between sample posting and receipt was 5 days (range 1–10). For culture, overall concordance for posted and fresh samples was 86% across the five targeted pathogens (ranging from 57 to 100% for different organisms), with no bias towards either sample type. For QPCR, overall concordance was 62% (range 39–84%), again with no bias towards fresh or posted samples. There were no significant differences in culture or QPCR for samples with short (≤3days) versus extended (≥7days) postal delays. Posting had no significant impact on pathogen abundance nor on microbiota characteristics. Posted sputum samples reliably reproduced culture-based and molecular microbiology of freshly collected samples, even after prolonged delays at ambient conditions. This supports use of posted samples during remote monitoring. [ABSTRACT FROM AUTHOR]

Growth curve measurements are one of the fundamental operations in microbiology and molecular biology. Modern microplate readers have the advantage of high-throughput and ease of automation. This allows for real-time monitoring of many small bacterial cultures and a significant decrease labor and materials costs. Using these devices, tens to hundreds of absorbance readings can be collected simultaneously for hundreds of samples. The small volume and the geometry of a well from a microplate implies that calibration and comparative studies must be performed before switching from one cultivation and growth monitoring system to another. Here, Escherichia coli XL1 Blue was used as a reference strain in order to test the feasibility of using a common, filter-based microplate reader for recording growth curves when performing antibiotic and toxicity testing. Two different cultivation conditions (flask cultivation and microplate cultivation) and three inoculum/media ratios were tested. The recorded growth curves indicated that the growth of the bacteria is much slower, the turbidity levels are lower, and reading are more variable in the microplate compared with the Erlenmayer flask. Several major technical difficulties must be carefully dealt with when measuring bacterial growth curves in microplates: efficiency of oxygenation that leads to slow growth rates and low densities, condensation on the microplate lead that leads to interference and evaporation of the growth media. [ABSTRACT FROM AUTHOR]

Two chronic osteomyelitis patients, a diabetic foot osteomyelitis patient and a fracture‐related infection patient, all with staphylococci‐positive microbiology, were examined to confirm the clinical relevance of bacterial invasion of the submicron osteocyte lacuna–canaliculi network (OLCN) in bone tissue. Based on immunohistochemistry and light microscopy both Staphylococcus aureus and Staphylococcus epidermidis were identified within the OLCN of all four patients. The findings consolidate that bacterial OLCN invasion is a clinically relevant part of osteomyelitis disease biology, which from experimental porcine infections, seems to be time depending. The microscopy pictures of the four patients significantly add to visualize the phenomenon of bacterial OLCN invasion. [ABSTRACT FROM AUTHOR]