Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

Microbial stir bars: Light-activated rotation of tethered bacterial cells to enhance mixing in stagnant fluids.

Microfluidics devices are gaining significant interest in biomedical applications. However, in a micron-scale device, reaction speed is often limited by the slow rate of diffusion of the reagents. Several active and passive micro-mixers have been fabricated to enhance mixing in microfluidic devices. Here, we demonstrate external control of mixing by rotating a rod-shaped bacterial cell. This rotation is driven by ion transit across the bacterial flagellar stator complex. We first measured the flow fields generated by rotating a single bacterial cell rotationally locked to rotate either clockwise (CW) or counterclockwise (CCW). Micro-particle image velocimetry (µPIV) and particle tracking velocimetry results showed that a bacterial cell of ∼ 2.75 µm long, rotating at 5.75 ± 0.39 Hz in a counterclockwise direction could generate distinct micro-vortices with circular flow fields with a mean velocity of 4.72 ± 1.67 µm/s and maximum velocity of 7.90 µm/s in aqueous solution. We verified our experimental data with a numerical simulation at matched flow conditions, which revealed vortices of similar dimensions and speed. We observed that the flow-field diminished with increasing z-height above the plane of the rotating cell. Lastly, we showed that we could activate and tune rotational mixing remotely using strains engineered with proteorhodopsin, where rotation could be activated by controlled external illumination using green laser light (561 nm).

Characterization of plasmids carrying blaCTX-M genes among extra-intestinal Escherichia coli clinical isolates in Ethiopia.

CTX-Ms are encoded by blaCTX-M genes and are widely distributed extended-spectrum ß-lactamases (ESBLs). They are the most important antimicrobial resistance (AMR) mechanism to ß-lactam antibiotics in the Enterobacteriaceae. However, the role of transmissible AMR plasmids in the dissemination of blaCTX-M genes has scarcely been studied in Africa where the burden of AMR is high and rapidly spreading. In this study, AMR plasmid transmissibility, replicon types and addiction systems were analysed in CTX-M-producing Escherichia coli clinical isolates in Ethiopia with a goal to provide molecular insight into mechanisms underlying such high prevalence and rapid dissemination. Of 100 CTX-Ms-producing isolates obtained from urine (84), pus (10) and blood (6) from four geographically distinct healthcare settings, 75% carried transmissible plasmids encoding for CTX-Ms, with CTX-M-15 being predominant (n = 51). Single IncF plasmids with the combination of F-FIA-FIB (n = 17) carried the bulk of blaCTX-M-15 genes. In addition, IncF plasmids were associated with multiple addiction systems, ISEcp1 and various resistance phenotypes for non-cephalosporin antibiotics. Moreover, IncF plasmid carriage is associated with the international pandemic E. coli ST131 lineage. Furthermore, several CTX-M encoding plasmids were associated with serum survival of the strains, but less so with biofilm formation. Hence, both horizontal gene transfer and clonal expansion may contribute to the rapid and widespread distribution of blaCTX-M genes among E. coli populations in Ethiopian clinical settings. This information is relevant for local epidemiology and surveillance, but also for global understanding of the successful dissemination of AMR gene carrying plasmids.

Atypical hemolytic uremic syndrome (aHUS) responsive to mycophenolate mofetil: A case report from Nepal.

Introduction: An atypical hemolytic uremic syndrome is an extremely rare and life-threatening thrombotic microangiopathy. This disorder is caused by dysregulation of the alternative pathway of the complement system in association with genetic abnormalities or the development of autoantibodies. However, 30-50% of patients do not have genetic or acquired mutations in the complement system. Case report: Patient presented with fever and periorbital swelling. She had anemia, thrombocytopenia, and deranged liver function tests. Urinalysis revealed hematuria and proteinuria. Antibody tests and genetic analysis were negative. Renal biopsy revealed findings suggestive of thrombotic microangiopathy with predominantly glomerular involvement. Thus, the diagnosis of Atypical Hemolytic Uremic Syndrome, immunofluorescence negative, genetic negative, and anti-complement negative was made. Discussion: This article reports a case of atypical hemolytic uremic syndrome in a child with negative genetic analysis and anti-complement factor H antibody, which was treated successfully on steroid and mycophenolate mofetil. Early diagnosis along with prompt treatment and close monitoring will lead to recovery from atypical Hemolytic Uremic Syndrome. Conclusion: Although HUS is generally associated with genetic abnormalities or a positive antibody test, some patients with HUS may present atypically with negative genetic analysis and antibody tests.

Type III secretion by Yersinia pseudotuberculosis is reliant upon an authentic N-terminal YscX secretor domain.

YscX was discovered as an essential part of the Yersinia type III secretion system about 20 years ago. It is required for substrate secretion and is exported itself. Despite this central role, its precise function and mode of action remain unknown. In order to address this knowledge gap, this present study refocused attention on YscX to build on the recent advances in the understanding of YscX function. Our experiments identified an N-terminal secretion domain in YscX promoting its secretion, with the first five codons constituting a minimal signal capable of promoting secretion of the signal less ß-lactamase reporter. Replacing the extreme YscX N-terminus with known secretion signals of other Ysc-Yop substrates revealed that the YscX N-terminal segment contains non-redundant information needed for YscX function. Further, both in cis deletion of the YscX N-terminus in the virulence plasmid and ectopic expression of epitope-tagged YscX variants again lead to stable YscX production but not type III secretion of Yop effector proteins. Mislocalisation of the needle components, SctI and SctF, accompanied this general defect in Yops secretion. Hence, a coupling exists between YscX secretion permissiveness and the assembly of an operational secretion system.

Antimicrobial Resistance Profiling and Molecular Epidemiological Analysis of Extended Spectrum ß-Lactamases Produced by Extraintestinal Invasive Escherichia coli Isolates From Ethiopia: The Presence of International High-Risk Clones ST131 and ST410 Revealed.

The treatment of invasive Escherichia coli infections is a challenge because of the emergence and rapid spread of multidrug resistant strains. Particular problems are those strains that produce extended spectrum ß-lactamases (ESBL's). Although the global characterization of these enzymes is advanced, knowledge of their molecular basis among clinical E. coli isolates in Ethiopia is extremely limited. This study intends to address this knowledge gap. The study combines antimicrobial resistance profiling and molecular epidemiology of ESBL genes among 204 E. coli clinical isolates collected from patient urine, blood, and pus at four geographically distinct health facilities in Ethiopia. All isolates exhibited multidrug resistance, with extensive resistance to ampicillin and first to fourth line generation cephalosporins and sulfamethoxazole-trimethoprim and ciprofloxacin. Extended spectrum ß-lactamase genes were detected in 189 strains, and all but one were positive for CTX-Ms ß-lactamases. Genes encoding for the group-1 CTX-Ms enzymes were most prolific, and CTX-M-15 was the most common ESBL identified. Group-9 CTX-Ms including CTX-M-14 and CTX-27 were detected only in 12 isolates and SHV ESBL types were identified in just 8 isolates. Bacterial typing revealed a high amount of strains associated with the B2 phylogenetic group. Crucially, the international high risk clones ST131 and ST410 were among the sequence types identified. This first time study revealed a high prevalence of CTX-M type ESBL's circulating among E. coli clinical isolates in Ethiopia. Critically, they are associated with multidrug resistance phenotypes and high-risk clones first characterized in other parts of the world.

Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics.

Many motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through membrane protein complexes known as stators, with the next most common ionic power source being sodium. Sodium-powered stators can be studied through the use of synthetic chimeric stators that combine parts of sodium- and proton-powered stator proteins. The most well studied example is the use of the sodium-powered PomA-PotB chimeric stator unit in the naturally proton-powered Escherichia coli. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria while varying the ionic composition of the media and thus the sodium-motive force available to drive this chimeric flagellar motor. We measured separation efficiencies at varying ionic concentrations and confirmed using fluorescence that our device delivered eightfold enrichment of the motile proportion of a mixed population. Furthermore, our results showed that we could select bacteria from reservoirs where sodium was not initially present. Overall, this technique can be used to implement the selection of highly motile fractions from mixed liquid cultures, with applications in directed evolution to investigate the adaptation of motility in bacterial ecosystems.

Knowledge, attitude and reported practice regarding donning and doffing of personal protective equipment among frontline healthcare workers against COVID-19 in Nepal: A cross-sectional study.

BACKGROUND: Coronavirus Disease 2019 (COVID-19) is a respiratory infection with a high rate of transmission primarily via airborne route and direct contact. Proper use of personal protective equipment (PPE) is a proven and effective way to prevent COVID-19 spread in healthcare settings. This study was done aiming to assess the knowledge, attitude, and reported practice, and identify the associated factors regarding donning and doffing of PPE among frontline healthcare workers in Nepal. METHODS: A cross-sectional study was conducted from 25th April to 30th July 2021 among 205 frontline healthcare workers of Nepal selected randomly from among the contacts of the investigators. A structured self-administered questionnaire prepared in google form was used as a study tool and shared via social media to the participants to obtain information on socio-demographic and workplace characteristics along with their knowledge, attitude, and reported practice regarding donning and doffing of PPE. RESULT: A total of 79.5% of participants had satisfactory knowledge while 75.6% had satisfactory practice scores regarding donning and doffing of PPE. Factors such as the profession of the participants (p-value = 0.048), their workplace (p-value = 0.005), provision of PPE at workplace (p-value = 0 .009), and availability of designated space at workplace for methodical donning and doffing of PPE (p-value = 0.010) were significantly associated with satisfactory knowledge score whereas availability of designated space at workplace for donning and doffing of PPE was significantly associated with good practice score (p-value = 0.009). CONCLUSION: This study demonstrated an overall good knowledge, attitude, and reported practice regarding donning and doffing of PPE among frontline healthcare workers in Nepal. However, the reported shortcomings like poor knowledge regarding the sequence of donning and doffing and corresponding flawed practice behaviors need to be addressed.

Microfluidic techniques for separation of bacterial cells via taxis.

The microbial environment is typically within a fluid and the key processes happen at the microscopic scale where viscosity dominates over inertial forces. Microfluidic tools are thus well suited to study microbial motility because they offer precise control of spatial structures and are ideal for the generation of laminar fluid flows with low Reynolds numbers at microbial lengthscales. These tools have been used in combination with microscopy platforms to visualise and study various microbial taxes. These include establishing concentration and temperature gradients to influence motility via chemotaxis and thermotaxis, or controlling the surrounding microenvironment to influence rheotaxis, magnetotaxis, and phototaxis. Improvements in microfluidic technology have allowed fine separation of cells based on subtle differences in motility traits and have applications in synthetic biology, directed evolution, and applied medical microbiology.

The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM.

The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

Heterologous Complementation Studies With the YscX and YscY Protein Families Reveals a Specificity for Yersinia pseudotuberculosis Type III Secretion.

Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenic Yersinia species, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither an yscX nor yscY null mutant of Yersinia is capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems of Pseudomonas aeruginosa (PscX and PscY), Aeromonas species (AscX and AscY), Vibrio species (VscX and VscY), and Photorhabdus luminescens (SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the native yscX in the near full-length yscX deletion and native yscY in the near full-length yscY deletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical for Yersinia type III secretion.

YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis.

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

Vibrio cholerae utilizes direct sRNA regulation in expression of a biofilm matrix protein.

Vibrio cholerae biofilms contain exopolysaccharide and three matrix proteins RbmA, RbmC and Bap1. While much is known about exopolysaccharide regulation, little is known about the mechanisms by which the matrix protein components of biofilms are regulated. VrrA is a conserved, 140-nt sRNA of V. cholerae, whose expression is controlled by sigma factor σE. In this study, we demonstrate that VrrA negatively regulates rbmC translation by pairing to the 5' untranslated region of the rbmC transcript and that this regulation is not stringently dependent on the RNA chaperone protein Hfq. These results point to VrrA as a molecular link between the σE-regulon and biofilm formation in V. cholerae. In addition, VrrA represents the first example of direct regulation of sRNA on biofilm matrix component, by-passing global master regulators.