An in silico hierarchal approach for drug candidate mining and validation of natural product inhibitors against pyrimidine biosynthesis enzyme in the antibiotic-resistant Shigella flexneri.

Shigella flexneri is the main causative agent of the communicable diarrheal disease, shigellosis. It is estimated that about 80-165 million cases and > 1 million deaths occur every year due to this disease. S. flexneri causes dysentery mostly in young children, elderly and immunocompromised patients, all over the globe. Recently, due to the emergence of S. flexneri antibiotic resistance strains, it is a dire need to predict novel therapeutic drug targets in the bacterium and screen natural products against it, which could eliminate the curse of antibiotic resistance. Therefore, in current study, available antibiotic-resistant genomes (n = 179) of S. flexneri were downloaded from PATRIC database and a pan-genome and resistome analysis was conducted. Around 5059 genes made up the accessory, 2469 genes made up the core, and 1558 genes made up the unique genome fraction, with 44, 34, and 13 antibiotic-resistant genes in each fraction, respectively. Core genome fraction (27% of the pan-genome), which was common to all strains, was used for subtractive genomics and resulted in 384 non-homologous, and 85 druggable targets. Dihydroorotase was chosen for further analysis and docked with natural product libraries (Ayurvedic and Streptomycin compounds), while the control was orotic acid or vitamin B13 (which is a natural binder of this protein). Dynamics simulation of 50 ns was carried out to validate findings for top-scored inhibitors. The current study proposed dihydroorotase as a significant drug target in S. flexneri and 4-tritriacontanone & patupilone compounds as potent drugs against shigellosis. Further experiments are required to ascertain validity of our findings.

The evolutionary history of Shigella flexneri serotype 6 in Asia.

Shigella flexneri serotype 6 is an understudied cause of diarrhoeal diseases in developing countries, and has been proposed as one of the major targets for vaccine development against shigellosis. Despite being named as S. flexneri, Shigella flexneri serotype 6 is phylogenetically distinct from other S. flexneri serotypes and more closely related to S. boydii. This unique phylogenetic relationship and its low sampling frequency have hampered genomic research on this pathogen. Herein, by utilizing whole genome sequencing (WGS) and analyses of Shigella flexneri serotype 6 collected from epidemiological studies (1987-2013) in four Asian countries, we revealed its population structure and evolutionary history in the region. Phylogenetic analyses supported the delineation of Asian Shigella flexneri serotype 6 into two phylogenetic groups (PG-1 and -2). Notably, temporal phylogenetic approaches showed that extant Asian S. flexneri serotype 6 could be traced back to an inferred common ancestor arising in the 18th century. The dominant lineage PG-1 likely emerged in the 1970s, which coincided with the times to most recent common ancestors (tMRCAs) inferred from other major Southeast Asian S. flexneri serotypes. Similar to other S. flexneri serotypes in the same period in Asia, genomic analyses showed that resistance to first-generation antimicrobials was widespread, while resistance to more recent first-line antimicrobials was rare. These data also showed a number of gene inactivation and gene loss events, particularly on genes related to metabolism and synthesis of cellular appendages, emphasizing the continuing role of reductive evolution in the adaptation of the pathogen to an intracellular lifestyle. Together, our findings reveal insights into the genomic evolution of the understudied Shigella flexneri serotype 6, providing a new piece in the puzzle of Shigella epidemiology and evolution.

Antibacterial, antifungal and enzymatic activities of azithromycin-heavy metal complexes: Newly synthesized and characterized.

As part of our continuous research to understand the interaction mechanism of drug and metallo-elements, heavy metal complexes of azithromycin (AZI) were synthesized with arsenic oxide, lead carbonate and silver chloride salts in molar ratio of 2: 1 (L: M). Synthesized heavy metal complexes have shown good percent yield and characterized through spectroscopic parameters including UV-Visible, TLC, FT-IR, NMR and elemental analysis (CHN). Spectroscopic characterization reveals the binding of ligand AZI with heavy metals in bi-dentate manner involving the hydroxide and 9a-NCH3 group of the aglycone ring of AZI. These newly synthesized heavy metal complexes were evaluated for their antimicrobial response against selected gram positive and gram negative organisms and antifungal species. It was noted that all newly synthesized complexes exhibits increased activity against B.subtilus whereas, AZI itself didn't show any activity, while synthesized complexes have low to moderate response against all the studied organisms. Complex A-M12 possess greater enzymatic response against both urease and alpha chymotrypsin among all the studied complexes. Results obtained were then statistically analyzed through one way ANOVA and Dunnett's test by using SPSS version 20.0 suggesting the significant response of complexes against selected organisms.

Novel amylomacins from seaweed-associated Bacillus amyloliquefaciens as prospective antimicrobial leads attenuating resistant bacteria.

The rise in antibiotic-resistant bacterial strains prompting nosocomial infections drives the search for new bioactive substances of promising antibacterial properties. The surfaces of seaweeds are rich in heterotrophic bacteria with prospective antimicrobial substances. This study aimed to isolate antibacterial leads from a seaweed-associated bacterium. Heterotrophic Bacillus amyloliquefaciens MTCC 12716 associated with the seaweed Hypnea valentiae, was isolated and screened for antimicrobial properties against drug-resistant pathogens. The bacterial crude extract was purified and three novel amicoumacin-class of isocoumarin analogues, 11'-butyl acetate amicoumacin C (amylomacin A), 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C (amylomacin B) and 11'-butyl amicoumacin C (amylomacin C) were isolated to homogeneity. The studied amylomacins possessed potential activities against Pseudomonas aeruginosa, vancomycin-resistant Enterococcus faecalis, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus, and Shigella flexneri with a range of minimum inhibitory concentration values from 0.78 to 3.12 µg/mL, although standard antibiotics ampicillin and chloramphenicol were active at 6.25-25 µg/mL. Noticeably, the amylomacin compound encompassing 4'-hydroxy-11'-methoxyethyl carboxylate amicoumacin C functionality (amylomacin B), displayed considerably greater antagonistic activities against methicillin-resistant S. aureus, vancomycin-resistant E. faecalis, Vibrio parahaemolyticus, Escherichia coli, and K. pneumoniae (minimum inhibitory concentration 0.78 µg/mL) compared to the positive controls and other amylomacin analogues. Antimicrobial properties of the amylomacins, coupled with the presence of polyketide synthase-I/non-ribosomal peptide synthetase hybrid gene attributed the bacterium as a promising source of antimicrobial compounds with pharmaceutical and biotechnological applications.

Multi-step Synthesis, Characterization and Photophysical Investigation of Novel Biologically Active Heterocyclic Chalcone (AECO).

Novel alkylated heterocyclic chalcone (E)-1-(2-(allyloxy)phenyl)-3-(9-ethyl-9H-carbazol-3-yl)prop-2-en-1-one (AECO) with extended π-bond was prepared by the multi-steps synthesis. The structure of the AECO was established by the spectroscopic technics and purity of the compound was confirmed by the elemental analysis. Physicochemical parameters of the AECO such as molar absorption coefficient, transition dipole moments, stokes shift, oscillator strength and fluorescence quantum yield were calculated in ten various solvents on the basis of polarity of the solvents to see the effect of the solvent with AECO. Interaction of the AECO chromophore with cationic CTAB and anionic SDS surfactants were determined by using the fluorescence spectroscopy techniques. The intensity of the florescence spectrum increase with increasing the concentrations of surfactants. This suggests that strong interaction occurs between AECO with surfactants and this interaction arise from electrostatic forces. So, AECO chromophore could be used as analysis to define the Critical Micelle Concentration (CMC) of the surfactants. In addition the in-vitro antibacterial active of novel heterocyclic chalcone agents four bacteria's strain were evaluated and result showed AECO is beater antibacterial agent against Gram-Negative Bacteria (E. coli and S. flexneri) as compare to the Gram Negative Bacteria with respected to the standard drug Tetracycline.

Effects of three essential oils and their nano-emulsions on Listeria monocytogenes and Shigella flexneri in Egyptian Talaga cheese.

Talaga cheese is a soft Egyptian cheese that has been associated with foodborne pathogens such as Listeria monocytogenes and Shigella flexneri. Essential oils (EOs) play a pivotal role in sustainably controlling foodborne diseases and as a potential preservative in soft cheeses. However, limited data is available comparing the antibacterial activity of EOs and their nano-emulsions (NEs) when inoculated into Talaga cheese. Therefore, this study aimed to examine the antibacterial activity of carvacrol, clove, and cumin EOs, in addition to their NEs, against L. monocytogenes (NCTC 13372/ATCC® 7644) and S. flexneri (ATCC®12022TW⁎) inoculated into laboratory-manufactured Egyptian Talaga cheese during refrigerated storage. The NEs had a Z-average diameter of 32.98 ± 29.75 nm, 45.2 ± 34.25 nm, and 50.23 ± 15.7 nm and a PDI of 0.326, 0.245, and 0.307 for carvacrol, clove, and cumin NEs, respectively. The flow of active functional groups of EOs and NEs as clarified by Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) showed the spherical-shaped droplet structure of the prepared NEs. The minimum inhibitory concentration (MIC) of all EOs and their NEs was 0.78% against L. monocytogenes and 1.56% against S. flexneri, while those of carvacrol EO and its NE were 0.78% against both microorganisms. By supplementation in cheese, NEs significantly reduced the counts of inoculated pathogens from 8.2 log10cfu/g to 1.5 log10cfu/g after 2 to 3 weeks compared to EOs, which reduced them after 4 to 5 weeks. The carvacrol NE showed excellent antibacterial activity with no cheese sensory impairment. It reduced L. monocytogenes by 99% (R%) after 7 days and after 3 weeks for S. flexneri at 0.78% concentration, while higher concentrations and a longer period were required for the other NEs to show an inhibitory effect. NEs showed a greater antimicrobial effect than their non-emulsified counterparts, especially when interacting with food items, and carvacrol NE at a low concentration (0.78%) demonstrated its efficacy as an antibacterial and natural food preservative.

Antibacterial and antishigellosis activity of Xylopia staudtii (engl. & diels), Annonaceae.

ETHNOPHARMACOLOGICAL RELEVANCE: Xylopia staudtii is a medicinal plant which fruits are traditionally used in western Cameroon as a spice in the preparation of soups known for their abdominal cramp relieving properties. Often identified as Xylopia africana, its bark is used in the treatment of dysentery in Mont Cameroun localities. This plant could therefore contain active ingredients against intestinal pathogens, including Shigella spp, which are responsible of the deathly dysenteric diarrhoea. AIM OF THE STUDY: This study aims to assess the efficacy of the hydroethanolic extract from Xylopia staudtii bark in immunodepressed mice infected with Shigella flexneri. MATERIALS AND METHODS: Qualitative detection of compounds in the crude extract was done using UPLC-DAD-(HR) ESI-MS analysis in an attempt to link the activity to the chemical composition. The MIC and the MBC of the extract was determined using broth dilution method. Shigellosis was induced by intraperitoneal administration of Shigella flexneri to immunodepressed mice pretreated with streptomycin. These infected mice were then treated with the extract (100, 200 and 400 mg/kg), and reference substances (ciprofloxacin and saline). During the 9 days of treatment, animal morphology, fecal pathology and deaths were recorded. At the end of the treatment period, blood and organs were collected from any surviving animals for hematological, biochemical and histopathological analyses. RESULTS: The extract was found to be significantly active, with a bactericidal effect against Shigella and a bacteriostatic effect against Escherichia coli. It was able to reduce and stop the faecal pathology caused by the infection in mice, as well as the rate of deaths which was brought to zero (0) in animal treated at 400 mg/kg. The bacteria load in faeces was reduced by 100% in animal treated at 400 mg/kg. Xylopia staudtii extract elicited anti-inflammatory properties by reducing MPO activity and Lcn2 intestinal level. It also prevents damages in the intestinal tissue and the shortening of colon which characterise Shigella infection. The serum level of ASAT, ALAT, bilirubin, urea and creatinine in animals treated with the extract was similar to those of normal animal used in the study. These activities of the plant may be due at least in part to the presence of ent-kauran type diterpens such as kaurenoic acid identified in the extract. CONCLUSION: These findings support the usage of Xylopia staudtii as an antimicrobial against bacillary dysentery, making this plant a potential candidate for the formulation of an improved standardized traditional medicine.

Correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri and the sul genes.

ABSTRACT: The aim of this study was to discuss the correlation between the sulfamethoxazole-trimethoprim resistance of Shigella flexneri (S. flexneri) and the antibiotic resistance genes sul1, sul2, and sul3 and SXT element.From May 2013 to October 2018, 102 isolates of S. flexneri were collected from the clinical samples in Jinan. The Kirby-Bauer (K-B) test was employed to determine the antibiotic susceptibility of the S. flexneri isolates. The antibiotic resistance rate was analyzed with the WHONET5.4 software. The isolates were subject to the PCR amplification of the sul genes (sul1, sul2, and sul3) and the SXT element. On the basis of the sequencing results, the correlation between the sulfamethoxazole-trimethoprim resistance of the S. flexneri isolates and the sul genes was analyzed.The antibiotic resistance rates of the 102 S. flexneri isolates to ampicillin, streptomycin, chloramphenicol, tetracycline, and sulfamethoxazole-trimethoprim were 90.2%, 90.2%, 88.2%, 88.2%, and 62.7%, respectively. The antibiotic resistance rates of these isolates to cefotaxime, ceftazidime, and ciprofloxacin varied between 20% and 35%. However, these isolates were 100% susceptible to cefoxitin. Positive fragments were amplified from 59.8% (61/102) of the 102 S. flexneri isolates, the sizes of the sul1 and sul2 genes being 338 bp and 286 bp, respectively. The sequence alignment revealed the presence of the sul1 and sul2 genes encoding for dihydrofolate synthase. The carrying rate of the sul1 gene was 13.7% (14/102), and that of the sul2 gene was 48.0% (49/102). No target gene fragments were amplified from the 3 isolates resistant to sulfamethoxazole-trimethoprim. The sul3 gene and SXT element were not amplified from any of the isolates. The testing and statistical analysis showed that the resistance of the S. flexneri isolates to sulfamethoxazole-trimethoprim correlated to the sul1 and sul2 genes.The acquired antibiotic resistance genes sul1 and sul2 were closely associated with the resistance of the 102 S. flexneri isolates to sulfamethoxazole-trimethoprim.

Evolutionary histories and antimicrobial resistance in Shigella flexneri and Shigella sonnei in Southeast Asia.

Conventional disease surveillance for shigellosis in developing country settings relies on serotyping and low-resolution molecular typing, which fails to contextualise the evolutionary history of the genus. Here, we interrogated a collection of 1,804 Shigella whole genome sequences from organisms isolated in four continental Southeast Asian countries (Thailand, Vietnam, Laos, and Cambodia) over three decades to characterise the evolution of both S. flexneri and S. sonnei. We show that S. sonnei and each major S. flexneri serotype are comprised of genetically diverse populations, the majority of which were likely introduced into Southeast Asia in the 1970s-1990s. Intranational and regional dissemination allowed widespread propagation of both species across the region. Our data indicate that the epidemiology of S. sonnei and the major S. flexneri serotypes were characterised by frequent clonal replacement events, coinciding with changing susceptibility patterns against contemporaneous antimicrobials. We conclude that adaptation to antimicrobial pressure was pivotal to the recent evolutionary trajectory of Shigella in Southeast Asia.

An in-vitro study on a novel six-phage cocktail against multi-drug resistant-ESBL Shigella in aquatic environment.

Shigella spp. are water-borne pathogens responsible for mild to severe cases bacilli dysentery all around the world known as Shigellosis. The progressively increasing of antibiotic resistance among Shigella calls for developing and establishing novel alternative therapeutic methods. The present study aimed to evaluate a novel phage cocktail of lytic phages against extended spectrum beta lactamase isolates of Shigella species in an aquatic environment. The phage cocktail containing six novel Shigella specific phages showed a broad host spectrum. The cocktail was very stable in aquatic environment. The cocktail resulted in about 99% decrease in the bacterial counts in the contaminated water by several species and strains of Shigella such as Shigella sonnei, Shigella flexneri and Shigella dysenteriae. Achieving such a high efficiency in this in-vitro study demonstrates a high potential for in-vivo and in-situ application of this phage cocktail as a bio-controlling agent against Shigella spp. contamination and infections.

Shigella sonnei: virulence and antibiotic resistance.

Shigella sonnei is the emerging pathogen globally, as it is the second common infectious species of shigellosis (bloody diarrhoea) in low- and middle-income countries (LMICs) and the leading one in developed world. The multifactorial processes and novel mechanisms have been identified in S. sonnei, that are collectively playing apart a substantial role in increasing its prevalence, while replacing the S. flexneri and other Gram-negative gut pathogens niche occupancy. Recently, studies suggest that due to improvement in sanitation S. sonnei has reduced cross-immunization from Plesiomonas shigelliodes (having same O-antigen as S. sonnei) and also found to outcompete the two major species of Enterobacteriaceae family (Shigella flexneri and Escherichia coli), due to encoding of type VI secretion system (T6SS). This review aimed to highlight S. sonnei as an emerging pathogen in the light of recent research with pondering aspects on its epidemiology, transmission, and pathogenic mechanisms. Additionally, this paper aimed to review S. sonnei disease pattern and related complications, symptoms, and laboratory diagnostic techniques. Furthermore, the available treatment reigns and antibiotic-resistance patterns of S. sonnei are also discussed, as the ciprofloxacin and fluoroquinolone-resistant S. sonnei has already intensified the global spread and burden of antimicrobial resistance. In last, prevention and controlling strategies are briefed to limit and tackle S. sonnei and possible future areas are also explored that needed more research to unravel the hidden mysteries surrounding S. sonnei.

Computational Identification of Essential Enzymes as Potential Drug Targets in Shigella flexneri Pathogenesis Using Metabolic Pathway Analysis and Epitope Mapping.

Shigella flexneri is a facultative intracellular pathogen that causes bacillary dysentery in humans. Infection with S. flexneri can result in more than a million deaths yearly and most of the victims are children in developing countries. Therefore, identifying novel and unique drug targets against this pathogen is instrumental to overcome the problem of drug resistance to the antibiotics given to patients as the current therapy. In this study, a comparative analysis of the metabolic pathways of the host and pathogen was performed to identify this pathogen's essential enzymes for the survival and propose potential drug targets. First, we extracted the metabolic pathways of the host, Homo sapiens, and pathogen, S. flexneri, from the KEGG database. Next, we manually compared the pathways to categorize those that were exclusive to the pathogen. Further, all enzymes for the 26 unique pathways were extracted and submitted to the Geptop tool to identify essential enzymes for further screening in determining the feasibility of the therapeutic targets that were predicted and analyzed using PPI network analysis, subcellular localization, druggability testing, gene ontology and epitope mapping. Using these various criteria, we narrowed it down to prioritize 5 novel drug targets against S. flexneri and one vaccine drug targets against all strains of Shigella. Hence, we suggest the identified enzymes as the best putative drug targets for the effective treatment of S. flexneri.

Secondary Structural Transformation of Bovine Lactoferricin Affects Its Antibacterial Activity.

Lactoferricin (Lfcin) is a potent antibacterial peptide derived from lactoferrin by pepsin hydrolysis. It was hypothesized that structural transformation of Lfcin could affect its antibacterial function through forming and breaking of intramolecular disulfide bond. To prove this hypothesis, bovine Lfcin (bLfcin) and its two derivatives, bLfcin with a disulfide bond (bLfcin DB) and bLfcin with a mutation C36G (bLfcin C36G), were synthesized, purified, and identified. The circular dichroism (CD) spectra of the peptides were detected in solutions with different ionic and hydrophobic strength. Then, the secondary structure contents of the peptides were calculated on the basis of the CD spectra. The antibacterial activity of the peptides against Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, Shigella flexneri ATCC 12022, and Staphylococcus aureus ATCC 25923 was evaluated. The results showed that bLfcin and bLfcin C36G had similar percentages of secondary structure in water, while bLfcin and bLfcin DB had similar ratios of secondary structure under less hydrophobic conditions. The synthetic peptides exhibited antibacterial activity against all the tested bacteria, except for S. aureus ATCC 25923. bLfcin demonstrated higher antibacterial activity compared with its derivatives. The results suggested that bLfcin could transform its structure under alterative ionic strengths and hydrophobic conditions, and the transformation of structures was beneficial to enhancing the antibacterial function.

Association of Serotype With Antimicrobial Resistance Patterns Among Shigella flexneri Isolates From Pakistan: The Importance of Serotype 2b.

BACKGROUND: Shigella flexneri is an emerging threat in low socioeconomic countries including Pakistan. No previous data is available on the association between S. flexneri serotypes and antimicrobial resistance in Pakistan. OBJECTIVES: The objective of the present study was to assess the association between serotypes and antimicrobial resistance patterns among S. flexneri isolated from clinical and nonclinical samples. METHODS: A total of 199 S. flexneri isolates were subjected to molecular serotyping and antibiotic resistance. RESULTS: The most prevalent S. flexneri serotype was 2b (38%) followed by 1b (24%), 7a (20%), 2a (11%), 1d (5%) and Y (2%). The phylogenetic reconstruction showed 12 clades among which the clades II, III, V, VIII, IX and XI have consisted of serotypes that were found both in human population and environment samples. A high level of multidrug resistance (MDR) was observed in serotype 2b (37.68%) followed by 1b (19.5%) and 7a (19.5%), 2a (11.5%), 1d (5%) and Y (2%). All isolates of serotype 2b showed high level of resistance to amoxicillin/clavulanic acid (100%) followed by quinolone (74.6%) and trimethoprim-sulfamethoxazole (54.6%). Interestingly, none of the serotype was resistant to piperacillin-tazobactam, imipenem and amikacin. The most frequently detected resistance genes among serotype 2b were blaOXA (100%) followed by qnrS (88%), cat (81%) and sul2 (63%). CONCLUSION: The most frequent S. flexneri serotype was 2b while 1d and Y was first time reported in Pakistan. High frequency of MDR serotypes of S. flexneri is a serious threat in diarrhea endemic regions and thus require urgent strategies for its continuous monitoring and prevention.

Newly Emerged Serotype 1c of Shigella flexneri: Multiple Origins and Changing Drug Resistance Landscape.

Bacillary dysentery caused by Shigella flexneri is a major cause of under-five mortality in developing countries, where a novel S. flexneri serotype 1c has become very common since the 1980s. However, the origin and diversification of serotype 1c remain poorly understood. To understand the evolution of serotype 1c and their antimicrobial resistance, we sequenced and analyzed the whole-genome of 85 clinical isolates from the United Kingdom, Egypt, Bangladesh, Vietnam, and Japan belonging to serotype 1c and related serotypes of 1a, 1b and Y/Yv. We identified up to three distinct O-antigen modifying genes in S. flexneri 1c strains, which were acquired from three different bacteriophages. Our analysis shows that S. flexneri 1c strains have originated from serotype 1a and serotype 1b strains after the acquisition of bacteriophage-encoding gtrIc operon. The maximum-likelihood phylogenetic analysis using core genes suggests two distinct S. flexneri 1c lineages, one specific to Bangladesh, which originated from ancestral serotype 1a strains and the other from the United Kingdom, Egypt, and Vietnam originated from ancestral serotype 1b strains. We also identified 63 isolates containing multiple drug-resistant genes in them conferring resistance against streptomycin, sulfonamide, quinolone, trimethoprim, tetracycline, chloramphenicol, and beta-lactamase. Furthermore, antibiotic susceptibility assays showed 83 (97.6%) isolates as either complete or intermediate resistance to the WHO-recommended first- and second-line drugs. This changing drug resistance pattern demonstrates the urgent need for drug resistance surveillance and renewed treatment guidelines.

Case report on a swift shift in uropathogens from Shigella flexneri to Escherichia coli: a thin line between bacterial persistence and reinfection.

BACKGROUND: Urinary tract infections (UTI) are mostly caused by bacteria. Urine cultures are usually a definitive measure to select the appropriate antibiotics for the elimination of a uropathogen and subsequent recovery from the infection. However, the preferred antibiotics as determined by urine culture and sensitivity may still not eliminate the infection and would require further examination to ascertain the cause of treatment failure which could be unresolved bacteriuria, bacterial persistence, immediate reinfection with a different uropathogen or misdiagnosis. CASE PRESENTATION: A 2-years 7 months-old female was admitted in the Regional hospital of Buea following persistent fever. An auto medication with amoxicillin was reported. Urinalysis was done on the first day and the sediment of the cloudy urine revealed many bacteria and few pus cells. Ceftriaxone was prescribed as empirical treatment and a request for urine and blood culture was made. Three days after admission, the temperature and CRP were 39.0 °C and 96 mg/l, respectively. The urine culture results (> 105 CFU/ml of Shigella flexneri sensitive to ofloxacin) were presented to the doctor on the 4th day of admission. Patient was put on ofloxacin. Three days after, the temperature (38.5 °C) and CRP (24 mg/l) were still elevated. The blood culture result came out negative. A second urine culture was requested which came back positive (> 105 CFU/ml of Escherichia coli resistant to ofloxacin and sensitive to meropenem and amikacin). Ofloxacin was discontinued and the patient put on meropenem and amikacin. The third urine culture recorded no significant growth after 48 h of incubation. The patient was discharged looking healthy once more with a normal body temperature. CONCLUSION: Antibiotics tailored towards the elimination of a particular bacterial species may as well provide a favorable environment for other bacterial species that are resistant to it in the course of treating a UTI episode. This apparent treatment failure may first of all require a second urine culture for confirmation rather than considering the possibilities of a misdiagnosis.

Phylogenetic Characterization Reveals Prevalent Shigella flexneri ST100 Clone in Beijing, China, 2005 to 2018.

Shigella flexneri is a major cause of bacillary dysentery in Beijing, China. The genetic features and population structure of locally circulating clones remained unclear. In this study, we sequenced the genomes of 93 S. flexneri isolates from patients in Beijing from 2005 to 2018. Phylogenetic analysis revealed a predominant lineage comprised of ST100 isolates that had acquired an extensive repertoire of antimicrobial resistance determinants. A rapid local expansion of the largest clade of this lineage began in 2008 and gradually resulted in the dominance of serotype 2a. Other clades showed substantial evidence of interregional spread from other areas of China. Another lineage consisting of ST18 isolates was also identified and appeared to have persisted locally for nearly 6 decades. These findings suggest that S. flexneri epidemics in Beijing were caused by both local expansion and interregional transmission.IMPORTANCE Beijing is the largest transportation hub in China, with a highly mobile population. Shigella flexneri is a major cause of bacillary dysentery in Beijing. However, little is known about the genetic features and population structure of locally circulating S. flexneri clones. Whole-genome sequencing of 93 S. flexneri isolates revealed that S. flexneri epidemics in Beijing were predominantly caused by an ST100 clone. Interregional spread, rapid local expansion, and acquirement of antimicrobial resistance determinants have cocontributed to the epidemics of this clone. Another ST18 clone was also identified and showed long-term colonization in Beijing. Our study provides comprehensive insights into the population structure and evolutionary history of S. flexneri in Beijing.

Novel Noncompetitive Type Three Secretion System ATPase Inhibitors Shut Down Shigella Effector Secretion.

Shigella is the causative agent of bacillary dysentery and is responsible for an estimated 165 million infections and 600,000 deaths annually. Like many Gram-negative pathogens, Shigella relies on a type three secretion system (T3SS) to initiate and sustain infection by directly injecting effector proteins into host cells. Protein secretion through the needle-like injectisome and overall Shigella virulence rely on the T3SS ATPase Spa47, making it a likely means for T3SS regulation and an attractive target for therapeutic small molecule inhibitors. Here, we utilize a recently solved 2.15 Å crystal structure of Spa47 to computationally screen 7.6 million drug-like compounds for candidates which avoid the highly conserved active site by targeting a distal, but critical, interface between adjacent protomers of the Spa47 homohexamer. Ten of the top inhibitor candidates were characterized, identifying novel Spa47 inhibitors that reduce in vitro ATPase activity by as much as 87.9 ± 10.5% with IC50's as low as 25 ± 20 µM and reduce in vivo Shigella T3SS protein secretion by as much as 94.7 ± 3.0%. Kinetic analyses show that the inhibitors operate through a noncompetitive mechanism that likely supports the inhibitors' low cytotoxicity, as they avoid off-target ATPases involved in either Shigella or mammalian cell metabolism. Interestingly, the inhibitors display nearly identical inhibition profiles for Spa47 and the T3SS ATPases EscN from E. coli and FliI from Salmonella. Together, the results of this study provide much-needed insight into T3SS ATPase inhibition mechanisms and a strong platform for developing broadly effective cross-pathogen T3SS ATPase inhibitors.

Ferulic Acid Inactivates Shigella flexneri through Cell Membrane Destructieon, Biofilm Retardation, and Altered Gene Expression.

Antibiotic resistance and capacity for biofilm formation of Shigella flexneri render previous prevention and control strategies minimally effective. Ferulic acid (FA) has been demonstrated to be useful due to its application in foods as an alternative natural preservative. However, information regarding the S. flexneri phenotype and molecular responses to FA exposure is limited. The present study investigated the effects of FA on S. flexneri planktonic growth and biofilm formation. The results demonstrated that the cell membrane of S. flexneri in planktonic growth mode exhibited irreversible destruction after FA exposure, as characterized by decreased cell viability, leakage of cytoplasmic constituents, accelerated adenosine triphosphate (ATP) consumption, cell membrane depolarization, and cellular morphological changes. FA significantly inhibited S. flexneri adhesion and biofilm formation at a working concentration (1/8 MIC) that almost did not inhibit planktonic growth. Transcriptomics profiling showed that the exposure to a subinhibitory concentration of FA dramatically altered gene expression in the S. flexneri biofilm, as a total of 169 differentially expressed genes (DEGs) were upregulated and 533 DEGs were downregulated, compared to the intact biofilm. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that the DEGs were mainly involved in pathways of ribosomes, ABC transporters, and the citrate cycle. Furthermore, we show that FA altered the transcription of S. flexneri genes associated with adhesion, transcriptional regulation, and the synthesis and transport of extracellular polymeric substances that contribute to biofilm formation. These data provide novel insights into S. flexneri behavioral responses to FA exposure and suggest that FA could effectively constrain S. flexneri and its biofilm formation.

Antiviral activity of the high-molecular-weight plant polysaccharides (Panavir®).

This short report is dedicated to the description of the wide antiviral and antibacterial activity of the immune-modulating agent Panavir®. Panavir® is a high-molecular-weight fraction of the polysaccharides extracted from the shoots of the Solanum tuberosum. It demonstrates activity against many types of viruses, including animal coronavirus and also against bacterial infections. These properties look very promising considering the COVID-19 epidemy and allow propose that Panavir® would be effective in the therapy of the SARS-CoV-2 infection.