Type IV pili of Enterobacteriaceae species.

Type IV pili (T4Ps) are surface filaments widely distributed among bacteria and archaea. T4Ps are involved in many cellular functions and contribute to virulence in some species of bacteria. Due to the diversity of T4Ps, different properties have been observed for homologous proteins that make up T4Ps in various organisms. In this review, we highlight the essential components of T4Ps, their functions, and similarities to related systems. We emphasize the unique T4Ps of enteric pathogens within the Enterobacteriaceae family, which includes pathogenic strains of Escherichia coli and Salmonella. These include the bundle-forming pilus (BFP) of enteropathogenic E. coli (EPEC), longus (Lng) and colonization factor III (CFA/III) of enterotoxigenic E. coli (ETEC), T4P of Salmonella enterica serovar Typhi, Colonization Factor Citrobacter (CFC) of Citrobacter rodentium, T4P of Yersinia pseudotuberculosis, a ubiquitous T4P that was characterized in enterohemorrhagic E. coli (EHEC), and the R64 plasmid thin pilus. Finally, we highlight areas for further study.

High throughput and targeted screens for prepilin peptidase inhibitors do not identify common inhibitors of eukaryotic gamma-secretase.

INTRODUCTION: Prepilin peptidases (PPP) are essential enzymes for the biogenesis of important virulence factors, such as type IV pili (T4P), type II secretion systems, and other T4P-related systems of bacteria and archaea. PPP inhibitors could be valuable pharmaceuticals, but only a few have been reported. Interestingly, PPP share similarities with presenilin enzymes from the gamma-secretase protease complex, which are linked to Alzheimer's disease. Numerous gamma-secretase inhibitors have been reported, and some have entered clinical trials, but none has been tested against PPP. OBJECTIVE: The objective of this study is to develop a high-throughput screening (HTS) method to search for inhibitors of PPP from various chemical libraries and reported gamma-secretase inhibitors. METHOD: More than 15,000 diverse compounds, including 13 reported gamma-secretase inhibitors and other reported peptidase inhibitors, were screened to identify potential PPP inhibitors. RESULTS: The authors developed a novel screening method and screened 15,869 compounds. However, the screening did not identify a PPP inhibitor. Nevertheless, the study suggests that gamma-secretase is sufficiently different from PPP that specific inhibitors may exist in a larger chemical space. CONCLUSION: The authors believe that the HTS method that they describe has numerous advantages and encourage others to consider its application in the search for PPP inhibitors.

Cryo-EM Structure of the Type IV Pilus Extension ATPase from Enteropathogenic Escherichia coli.

Type 4 pili (T4P) are retractable surface appendages found on numerous bacteria and archaea that play essential roles in various microbial functions, including host colonization by pathogens. An ATPase is required for T4P extension, but the mechanism by which chemical energy is transduced to mechanical energy for pilus extension has not been elucidated. Here, we report the cryo-electron microscopy (cryo-EM) structure of the BfpD ATPase from enteropathogenic Escherichia coli (EPEC) in the presence of either ADP or a mixture of ADP and AMP-PNP. Both structures, solved at 3 Å resolution, reveal the typical toroid shape of AAA+ ATPases and unambiguous 6-fold symmetry. This 6-fold symmetry contrasts with the 2-fold symmetry previously reported for other T4P extension ATPase structures, all of which were from thermophiles and solved by crystallography. In the presence of the nucleotide mixture, BfpD bound exclusively AMP-PNP, and this binding resulted in a modest outward expansion in comparison to the structure in the presence of ADP, suggesting a concerted model for hydrolysis. De novo molecular models reveal a partially open configuration of all subunits where the nucleotide binding site may not be optimally positioned for catalysis. ATPase functional studies reveal modest activity similar to that of other extension ATPases, while calculations indicate that this activity is insufficient to power pilus extension. Our results reveal that, despite similarities in primary sequence and tertiary structure, T4P extension ATPases exhibit divergent quaternary configurations. Our data raise new possibilities regarding the mechanism by which T4P extension ATPases power pilus formation. IMPORTANCE Type 4 pili are hairlike surface appendages on many bacteria and archaea that can be extended and retracted with tremendous force. They play a critical role in disease caused by several deadly human pathogens. Pilus extension is made possible by an enzyme that converts chemical energy to mechanical energy. Here, we describe the three-dimensional structure of such an enzyme from a human pathogen in unprecedented detail, which reveals a mechanism of action that has not been seen previously among enzymes that power type 4 pilus extension.

Landmark Discoveries and Recent Advances in Type IV Pilus Research.

Type IV pili (T4P) are retractable multifunctional nanofibers present on the surface of numerous bacterial and archaeal species. Their importance to microbiology is difficult to overstate. The scientific journey leading to our current understanding of T4P structure and function has included many innovative research milestones. Although multiple T4P reviews over the years have emphasized recent advances, we find that current reports often omit many of the landmark discoveries in this field. Here, we attempt to highlight chronologically the most important work on T4P, from the discovery of pili to the application of sophisticated contemporary methods, which has brought us to our current state of knowledge. As there remains much to learn about the complex machine that assembles and retracts T4P, we hope that this review will increase the interest of current researchers and inspire innovative progress.

Psychiatric Disorders in Adolescents Attending Psychiatry Outpatient Department in a Tertiary Care Hospital: A Descriptive Cross-sectional Study.

INTRODUCTION: Adolescence is the time when most mental illness begins most of the time these problems are overlooked leading to increased morbidity and mortality. The study is undertaken to find out the prevalence of psychiatric disorders in adolescents attending psychiatry outpatient department in a tertiary hospital. METHODS: A descriptive cross-sectional study was done from 1st December 2019 to 29th November 2020 at a tertiary care centre of Kathmandu. Ethical approval (Ref No.: 012-076/077) was taken from the Institution Review Committee. A semi-structured proforma was used for the socio-demographic profile of patients and International Classification of Diseases-10 was used to make the diagnosis. Data were analyzed using Statistical Package for the Social Sciences version 16. RESULTS: A total of 174 adolescents were included in the study. Out of the total cases 141 (81.03%) (75.1-86.8 at 95% Confidence Interval) were diagnosed with psychiatric disorders, among them 73 (51.77%) were males and 68 (48.23%) were females. The common diagnoses were anxiety disorders 63 (36.20%), mood disorders 34 (19.54%), psychotic disorders 26 (14.94%), substance use disorders 8 (4.59%), non-organic sleep disorders 5 (2.87%), behavioral and emotional disorders 3 (1.72%) and mental retardation 2 (1.149%). CONCLUSIONS: Prevalence of psychiatric illnesses is high in the adolescent population compared to the similar studies.

Draft genome sequences of concurrent Escherichia coli blood and fecal isolates from a patient with bacteremia and diarrhea belie BioFire-based detection of fecal enteropathogenic E. coli.

The BioFire FilmArray® Gastrointestinal panel is a multiplex PCR assay widely used to determine the etiology of infectious gastroenteritis directly from stool specimens. Recently a positive BioFire result for fecal enteropathogenic Escherichia coli (EPEC) was reported by a clinical microbiology laboratory for an adult patient with diarrhea and bacteremia. Since EPEC infrequently infects adults and rarely causes bacteremia, we isolated fecal E. coli and characterized the patient's blood and fecal E. coli isolates. Draft genome sequencing using a combination of methods indicated that the blood and fecal strains are virtually identical, are from sequence type 963 (phylogroup D) and exhibit neither the virulence genes characteristic of EPEC and extraintestinal pathogenic E. coli (ExPEC) nor classic EPEC-associated phenotypes. These findings support a gut source for the patient's bacteremia but exclude EPEC as the causative organism, and suggest that results of multiplex PCR assays from complex samples can be misleading, and should be interpreted with caution when they are discordant with clinical information. BioProject accession numbers for strains MVAST5574 and MVAST5635 genomes are PRJNA611789 and PRJNA611804, respectively.

Cystic lymphangioma of the stomach with marked reactive changes: a rare cause of gastric outlet obstruction in adult.

Cystic lymphangiomas are benign lymphatic tumours which usually affect the paediatric population and are predominantly located in the head and neck region. Its occurrence during adulthood and an intra-abdominal location are both extremely uncommon. Clinically and radiologically, these lesions often mimic malignancy. Infrequently, these tumours can undergo degenerative and reactive changes obscuring the diagnostic features. We describe hereby an anecdote of cystic lymphangioma with marked reactive changes presenting with the features of gastric outlet obstruction in an adult patient.

Prevalence of Depression in Patients with Chronic Kidney Disease Stage 5 on Hemodialysis at a Tertiary Care Center.

INTRODUCTION: Co-morbid depression impacts negatively on quality of life in Chronic Kidney Disease patients. This study was done to calculate prevalence of depression in patients on hemodialysis (HD) using Patient Health Questionnaire-9, Hamilton Rating Scale for Depression-17 and International Classification of Disease-10 classification of mental and behavioural disorders. METHODS: It was descriptive cross-sectional study conducted from November 2017 till June 2018. Ethical approval was taken from Ethical Review Board, Nepal Health Research Council. Informed and written consent was taken. Patients undergoing hemodialysis at Nepal Medical College for more than 3 months duration were included in study. Patients on hemodialysis were asked to fill validated Nepali translated version of Patient Health Questionnaire-9. Psychiatrist administered Max Hamilton Rating scale for diagnosis and categorization of depression and confirmed depression based on International Classification of Disease-10. Statistical Package for Social Sciences version 20 was used for statistical analysis. RESULTS: The study was done among 100 patients. Prevalence of depression was 78 (78%) using Patient Health Questionnaire-9 and 65 (65%) using Max Hamilton Rating scale and 51 (51%) using International Classification of Disease -10. Mean depression in males using Patient Health Questionnaire -9 was 7±4.33 and in females was 11.04±5.90. Median age was 47.5 years. The most common symptom was fatigue among 82 (82%). CONCLUSIONS: There is a high prevalence of depression in patients with Chronic Kidney Disease stage 5 on hemodialysis compared to general population.

Bacillus mangrovi sp. nov., isolated from a sediment sample from a mangrove forest.

A facultatively anaerobic, endospore forming, alkali-tolerant, Gram-stain-positive, motile, rod-shaped bacterium, designated strain AK61T, was isolated from a sediment sample collected from Coringa mangrove forest, India. Colonies were circular, 1.5 mm in diameter, shiny, smooth, yellowish and convex with entire margins after 48 h growth at 30 °C. Growth occurred at 15-42 °C, with 0-3 % (w/v) NaCl and at pH 6-9. AK61T was positive for amylase activity and negative for oxidase, catalase, aesculinase, caseinase, cellulase, DNase, gelatinase, lipase and urease activities. The fatty acids were dominated by branched types with iso- and anteiso- saturated fatty acids with a high abundance of iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0 and iso-C16 : 0; the cell-wall peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid; and MK-7 was the major menaquinone. DNA-DNA hybridization between AK61T and Bacillus indicus MTCC 4374T and between AK61T and Bacillus indicus KCTC 3880 showed relatedness of 37.99 and 33.32 % respectively. The DNA G+C content of AK61T was 44 mol%. The results of a blast sequence similarity search based on 16S rRNA gene sequences indicated that Bacillus cibi and Bacillus indicus were the nearest phylogenetic neighbours, with a pair-wise sequence similarity of 97.69 and 97.55 % respectively. The results of phylogenetic analysis indicated that AK61T was clustered with Bacillus idriensis and Bacillus indicus. On the basis of its phenotypic characteristics and phylogenetic inference, AK61T represents a novel species of the genus Bacillus, for which the name Bacillus mangrovi sp. nov. is proposed. The type strain is AK61T (=JCM 31087T=MTCC 12015T=KCTC 33872T).

Characterization of heat induced spherulites of lysozyme reveals new insight on amyloid initiation.

Here, we report results obtained during our experiments to visualize how heat transforms globular protein, lysozyme into building block of ß-amyloids. Light scattering experiments showed formation of lower order associated species around 50-70 °C followed by rapid cooperativity to ß-amyloid fibrils. Interestingly, crystallization drops set at higher temperatures either led to aggregates or spherulites. The latter possess an amorphous ß-fibril rich core with thin crystalline needles projecting outwards. Diffraction of the crystalline outgrowths revealed novel dimers and trimers of lysozyme where individual chains were similar to monomer with marginal gain in ß-sheet content. Importantly, analysis of Amide I stretching frequencies showed that protein loses its secondary structure at temperatures higher than where we obtained crystals followed by rapid gain in ß-sheet content. Interestingly, attempts to use the needles as seeds for more crystals led to "broom-like" fibril formations at the ends. Further, aggregation inhibitors like arginine and benzyl alcohol completely obliterated spherulites formation during crystallization. Refinement of crystals of lysozyme in presence of these molecules showed these small molecules bind to the interfaces of heat associated dimers and trimers. Overall our work concludes that heat induced weakly associated structures of lysozyme are the first step towards its amyloid formation.

Rhodococcus lactis sp. nov., an actinobacterium isolated from sludge of a dairy waste treatment plant.

A Gram-stain-positive, non-motile and aerobic bacterium, designated strain DW151BT, was isolated from a sludge sample of a dairy industry effluent treatment plant. 16S rRNA gene sequence analysis of strain DW151BT placed it within the genus Rhodococcus. It displayed significant similarity with recognized species of the genus: Rhodococcus pyridinivorans PDB9T (98.8 %), Rhodococcus gordoniae W 4937T (98.6 %), Rhodococcus rhodochrous DSM 43241T (98.5 %) and Rhodococcus artemisiae YIM 65754T (97.5 %). However, strain DW151BT differed from phylogenetically closely related species in various phenotypic properties. The cellular polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as major lipids, MK-8(H2) was the major menaquinone and meso-diaminopimelic acid was the cell-wall peptidoglycan. The fatty acid profile consisted of C16 : 0, C18 : 1cis9 and C16 : 1cis9 as main components. The presence of C16 : 0 and diphosphatidylglycerol as major fatty acid and polar lipid, respectively, was in accordance with chemotaxonomic markers of the genus Rhodococcus. The DNA G+C content of strain DW151BT was 69.9 mol%, a value within the limits reported for the members of this genus. Furthermore, strain DW151BT showed low similarity at the whole genome level in DNA-DNA hybridization experiments with phylogenetically closely related strains. Considering the low similarity at the genome level and differences in phenotypic properties, strain DW151BT is considered to represent a novel species of the genus Rhodococcus, for which the name Rhodococcus lactis sp. nov. is proposed. The type strain is DW151BT ( = MTCC 12279T = DSM 45625T).

The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin, conceals a human defensin-like structural module.

The growing emergence of antibiotic-resistant bacteria has led to the exploration of naturally occurring defense peptides as antimicrobials. In this study, we found that laterosporulin (LS), a class IId bacteriocin, effectively kills active and nonmultiplying cells of both Gram-positive and Gram-negative bacteria. Fluorescence and electron microscopy suggest that growth inhibition occurs because of increased membrane permeability. The crystal structure of LS at 2.0 Å resolution reveals an all-ß conformation of this peptide, with four ß-strands forming a twisted ß-sheet. All six intrinsic cysteines are intramolecularly disulfide-bonded, with two disulfides constraining the N terminus of the peptide and the third disulfide crosslinking the extreme C terminus, resulting in the formation of a closed structure. The significance of disulfides in maintaining the in-solution peptide structure was confirmed by CD and fluorescence analyses. Despite a low overall sequence similarity, LS has disulfide connectivity [C(I)-C(V), C(II)-C(IV), and C(III)-C(VI)] like that of ß-defensins and a striking architectural similarity with α-defensins. Therefore LS presents a missing link between bacteriocins and mammalian defensins, and is also a potential antimicrobial lead, in particular against nonmultiplying bacteria.

A non-pediocin low molecular weight antimicrobial peptide produced by Pediococcus pentosaceus strain IE-3 shows increased activity under reducing environment.

BACKGROUND: Species of the genus Pediococcus are known to produce antimicrobial peptides such as pediocin-like bacteriocins that contain YGNGVXC as a conserved motif at their N-terminus. Until now, the molecular weight of various bacteriocins produced by different strains of the genus Pediococcus have been found to vary between 2.7 to 4.6 kD. In the present study, we characterized an antimicrobial peptide produced by P. pentosaceus strain IE-3. RESULTS: Antimicrobial peptide was isolated and purified from the supernatant of P. pentosaceus strain IE-3 grown for 48 h using cation exchange chromatography and reversed-phase high-performance liquid chromatography (RP-HPLC) techniques. While MALDI-TOF MS experiments determined the precise molecular mass of the peptide to be 1701.00 Da, the de novo sequence (APVPFSCTRGCLTHLV) of the peptide revealed no similarity with reported pediocins and did not contain the YGNGVXC conserved motif. Unlike pediocin-like bacteriocins, the low molecular weight peptide (LMW) showed resistance to different proteases. Moreover, peptide treated with reducing agent like dithiothreitol (DTT) exhibited increased activity against both Gram-positive and Gram-negative test strains in comparison to native peptide. However, peptide treated with oxidizing agent such as hydrogen peroxide (H2O2) did not show any antimicrobial activity. CONCLUSION: To our knowledge this is the lowest molecular weight peptide produced by members of the genus Pediococcus. The low molecular weight peptide shared amino acid arrangement with N-terminal sequence of Class IIa, pediocin-like bacteriocins and showed increased activity under reducing conditions. Antimicrobial peptides active under reduced conditions are valuable for the preservation of processed foods like meat, dairy and canned foods where low redox potential prevails.

An unusual case of gastric outlet obstruction caused by tuberculosis: challenges in diagnosis and treatment.

Gastroduodenal tuberculosis (GDTB) is rare in the West. Its presentation can be non-specific and often mimics other more common conditions such as peptic ulcer disease, malignancy and Crohn's disease. Our case describes a 33-year-old Indian immigrant who presented with a 3-year history of dyspepsia and underwent balloon dilation for gastric outlet obstruction (GOO). While biopsies from the duodenum revealed only non-caseating granuloma, a high index of suspicion was maintained and colonoscopy, performed despite the absence of lower gastrointestinal symptoms, revealed a single discrete nodular and ulcerated area in the proximal transverse colon; this eventually grew Mycobacterium tuberculosis. Our patient avoided undergoing major surgery and was successfully treated with balloon dilation and antitubercular medication. We highlight the importance of having a concerted, proactive approach to diagnosis. We discuss the therapeutic challenges involving this rare condition and explain the rationale for high-dose antisecretory therapy.

Characterization of two antimicrobial peptides produced by a halotolerant Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample.

A bacterial strain producing two antimicrobial peptides was isolated from a rhizosphere soil sample and identified as Bacillus subtilis based on both phenotypic and 16S rRNA gene sequence phylogenetic analysis. It grew optimally up to 14% NaCl and produced antimicrobial peptide within 24 h of growth. The peptides were purified using a combination of chemical extraction and chromatographic techniques. The MALDI-TOF analysis of HPLC purified fractions revealed that the strain SK.DU.4 secreted a bacteriocin-like peptide with molecular mass of 5323.9 Da and a surface-active lipopeptide (m/z 1056 Da). The peptide mass fingerprinting of low-molecular-weight bacteriocin exhibited significant similarity with stretches of secreted lipoprotein of Methylomicrobium album BG8 and displayed 70% sequence coverage. MALDI MS/MS analysis elucidated the lipopeptide as a cyclic lipopeptide with a ß-hydroxy fatty acid linked to Ser of a peptide with seven α-amino acids (Asp-Tyr-Asn-Gln-Pro-Asn-Ser) and assigned it to iturin-like group of antimicrobial biosurfactants. However, it differed in amino acid composition with other members of the iturin family. Both peptides were active against Gram-positive bacteria, suggesting that they had an additive effect.

Genome sequence of Pediococcus pentosaceus strain IE-3.

We report the 1.8-Mb genome sequence of Pediococcus pentosaceus strain IE-3, isolated from a dairy effluent sample. The whole-genome sequence of this strain will aid in comparative genomics of Pediococcus pentosaceus strains of diverse ecological origins and their biotechnological applications.

Identification, purification and characterization of laterosporulin, a novel bacteriocin produced by Brevibacillus sp. strain GI-9.

BACKGROUND: Bacteriocins are antimicrobial peptides that are produced by bacteria as a defense mechanism in complex environments. Identification and characterization of novel bacteriocins in novel strains of bacteria is one of the important fields in bacteriology. METHODOLOGY/FINDINGS: The strain GI-9 was identified as Brevibacillus sp. by 16 S rRNA gene sequence analysis. The bacteriocin produced by strain GI-9, namely, laterosporulin was purified from supernatant of the culture grown under optimal conditions using hydrophobic interaction chromatography and reverse-phase HPLC. The bacteriocin was active against a wide range of Gram-positive and Gram-negative bacteria. MALDI-TOF experiments determined the precise molecular mass of the peptide to be of 5.6 kDa and N-terminal sequencing of the thermo-stable peptide revealed low similarity with existing antimicrobial peptides. The putative open reading frame (ORF) encoding laterosporulin and its surrounding genomic region was fished out from the draft genome sequence of GI-9. Sequence analysis of the putative bacteriocin gene did not show significant similarity to any reported bacteriocin producing genes in database. CONCLUSIONS: We have identified a bacteriocin producing strain GI-9, belonging to the genus Brevibacillus sp. Biochemical and genomic characterization of laterosporulin suggests it as a novel bacteriocin with broad spectrum antibacterial activity.

Genome sequence of Brevibacillus laterosporus strain GI-9.

We report the 5.18-Mb genome sequence of Brevibacillus laterosporus strain GI-9, isolated from a subsurface soil sample during a screen for novel strains producing antimicrobial compounds. The draft genome of this strain will aid in biotechnological exploitation and comparative genomics of Brevibacillus laterosporus strains.

Fulvivirga imtechensis sp. nov., a member of the phylum Bacteroidetes.

A novel, Gram-staining-negative, yellow-coloured, rod-shaped, obligately aerobic, non-motile bacterium, designated strain AK7(T), was isolated from seawater collected on the coast at Visakhapatnam, Andhra Pradesh, India. The predominant fatty acids of the novel strain were iso-C(15 : 0), iso-C(15 : 0) 3-OH, C(16 : 1)ω5c, iso-C(17 : 0) 3-OH and summed features 3 (C(16 : 1)ω7c and/or iso-C(15 : 0) 2-OH) and 4 (iso-C(17 : 1) I and/or anteiso-C(17 : 1) B). The major respiratory quinone was MK-7 and the polar lipid profile comprised phosphatidylethanolamine, two unidentified aminolipids and four other unidentified lipids. In phylogenetic analysis based on 16S rRNA gene sequences, strain AK7(T) appeared most closely related to Fulvivirga kasyanovii KMM 6220(T) (95.9 % sequence similarity), a member of the family Flammeovirgaceae in the phylum Bacteroidetes. The genomic DNA G+C content of strain AK7(T) was 55.1 mol%. Based on the morphological, biochemical, physiological, chemotaxonomic and phylogenetic evidence, strain AK7(T) represents a novel species of the genus Fulvivirga for which the name Fulvivirga imtechensis sp. nov. is proposed. The type strain is AK7(T) (= MTCC 11053(T) = JCM 17390(T)).