Seroprevalence of brucellosis among animal handlers in West Bengal, India: an occupational health study.

Brucellosis is a highly contagious zoonotic disease and a major human health problem worldwide. Due to its ways of transmission, direct or indirect contact with infected animals or their contaminated biological products, the disease exhibits strong occupational association with animal handlers comprising a significant population at risk. This study was undertaken to estimate the seroprevalence of brucellosis in animal handlers and to understand the epidemiological and serological aspects of the same. The animal handlers from the state of West Bengal, India were included in this study. It was a prospective and observational cohort study from November 2021 to March 2022. A total of 669 sera samples were collected from animal handlers and tested using various serological tests for Brucella antibodies. All serum samples were tested using the Rose Bengal plate test (RBPT), standard tube agglutination test (STAT), and enzyme-linked immunosorbent assay (ELISA). 106 (15.8%) patients were diagnosed with brucellosis among the total number of patients tested. Most of the patients affected with brucellosis belonged to the age group 51-60 years (23.5%). The seropositivity rate in male animal handlers was higher than female animal handlers in this study. More studies are needed to understand the occupational association of this disease. Awareness programs, safe livestock practices, and prevention of the disease by timely diagnosis must be implemented in order to control human brucellosis.

Current Trends in Antimicrobial Resistance Patterns in Bacterial Pathogens among Adult and Pediatric Patients in the Intensive Care Unit in a Tertiary Care Hospital in Kolkata, India.

Nosocomial infections by multidrug-resistant (MDR) bacteria are among the main causes of morbidity and death in patients hospitalized in intensive care units (ICUs) worldwide. Antibiotic resistance has become a major concern for treating the patients with nosocomial infections. The aim of this study was to describe the antibiotic resistance patterns of pathogens causing infections in adult and pediatric patients in the ICUs of a tertiary care hospital in Kolkata, India. A cross-sectional, retrospective study was conducted from January 2022 to October 2022 on a total of 139 adult and 146 pediatric patients. Depending on clinical symptoms of the patients, samples were collected and subjected to antibiotic sensitivity testing. The culture and sensitivity pattern of clinical isolates from blood, urine, sputum, endotracheal tube (ET) aspirate, and central line catheter insertion site swabs were analyzed. A total of 695 and 556 specimens were obtained from adult and pediatric ICU, respectively. Culture positivity rate among adults and pediatric patients were 37% and 40%, respectively. The most commonly isolated organisms were Gram-negative Enterobacterales and non-fermenters. Most of the bacterial isolates showed very high resistance against multiple antibiotics. Escherichia coli from adult and pediatricpatients were found to be resistant to second generation cephalosporins (95% and 96%, respectively), beta-lactams (95% and 63%, respectively), fluoroquinolones (95% and 81%, respectively), and cotrimoxazole (85% and 78%, respectively). Klebsiella spp. from adult patients were found to be resistant to aminoglycosides (75%), second generation cephalosporins (100%), beta-lactams (94%), fluoroquinolones (92%), carbapenems (88%), and cotrimoxazole (83%). Proteus spp., Acinetobacter baumannii, and Pseudomonas spp. werefound to be resistant to multiple antibiotics. Enterococcus spp. from ICUs showed more than 90% resistance against ampicillin and more than 75% resistance against fluoroquinolones. MDR bacterial infections are increasing in both adult and pediatric ICUs, leading to significant therapeutic challenges. A frequent study of antimicrobial resistance patterns is imperative for antibiotic stewardshipin combatting the deadly effect of the MDR bacteria in critically ill patients.

Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli.

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32 °C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

Biochemical and biophysical analysis of the interaction of a recombinant form of Staphylococcus aureus enolase with plasminogen.

Aim: Pathogenic invasion of Staphylococcus aureus is critically dependent on host plasminogen activation. Materials & methods: The pathophysiological implications of the interactions between S. aureus recombinant enolase and host plasminogen were investigated. The effects of mutation and small synthetic peptide inhibitors on interactions were assessed. Results: In vitro, the S. aureus recombinant enolase exists as a catalytically active fragile octamer and a robust dimer. The dimer interacts with the host plasminogen on the S. aureus surface. Conclusion: The interaction of host plasminogen and S. aureus enolase might mediate bacterial adherence to the host, activate the plasminogen with the help of plasminogen activators and prevent α2-antiplasmin-mediated inhibition of plasmin. Incorporating mutant and synthetic peptides inhibited the interactions and their associated pathophysiological consequences.

Changing Paradigms in Antibiotic Resistance in Salmonella Species with Focus on Fluoroquinolone Resistance: A 5-Year Retrospective Study of Enteric Fever in a Tertiary Care Hospital in Kolkata, India.

Enteric fever, a potentially fatal multisystem disease that is caused by Salmonella enterica serovar Typhi and Paratyphi, poses a significant risk in low- and middle-income countries. A retrospective study to understand the prevalence and evolving patterns of antibiotic resistance in Salmonella Typhi and Paratyphi was undertaken from June 2017 to June 2022. A total of 4051 blood samples were collected from patients attending inpatient and outpatient departments of the School of Tropical Medicine (Kolkata, India) hospital. Blood samples were cultured, and culture positive samples were further processed for identification using conventional and automated systems. Antibiotic susceptibility test was performed using both the Kirby-Bauer disc diffusion method and VITEK2 (bioMerieux). Forty-five (1.1%) Salmonella species were isolated among the number of total (n = 4051) samples that were tested. Out of the 45 Salmonella isolates, 35 were Salmonella Typhi (77.77%) and 10 were Salmonella Paratyphi A (22.23%). We found pronounced fluoroquinolone resistance of 100% in the recent years (2019-2022) in both of the S. Typhi and S. Paratyphi A isolates. We found that 1 Salmonella Typhi and 2 Salmonella Paratyphi A isolates were resistant against multiple antibiotics (cefixime, ceftriaxone, ciprofloxacin and nalidixic acid), and 1 multidrug-resistant (MDR) Salmonella Paratyphi A isolate was found in a recent study year (2020) and it showed resistance against different classes of antibiotics (cephalosporins, fluoroquinolones and carbapenems). There was no resistance that was detected to the 3rd generation cephalosporins in the final years of the study. The emergence of Salmonella isolates that are resistant to multiple antibiotics poses a serious health problem. The antimicrobial resistance patterns that were detected in the study thus warrant further studies to understand the antibiotic susceptibility and resistance pattern of Salmonella against the major classes of antibiotics.

A coumarin coupled tetraphenylethylene based multi-targeted AIEgen for cyanide ion and nitro explosive detection, and cellular imaging.

A coumarin coupled tetraphenylethylene based AIEgen (TPE-Lac) with an intense greenish-yellow emission has been synthesized and utilized for multipurpose sensing and imaging applications. TPE-Lac acts as a sensitive sensor for the detection of cyanide ions (CN-) with an immediate turn-off response in the presence of many other interfering cations and anions. The limit of detection (LOD) was as low as 33 nM, which is well below the permissible limit set by the World Health Organization (WHO). Cyanide detection in the solid phase was successfully demonstrated by drop-casting the solution of the TPE-Lac probe on TLC plates and measuring and analysing the fluorescence response by ImageJ analysis. TPE-Lac was further employed in the detection of explosive nitroaromatics in solution and solid phases. Also, TPE-Lac was found suitable as an imaging agent and could easily percolate into live H520 cells giving bright fluorescence from the intra-cellular region. Easy and cost-effective synthesis, fast response and low LODs are some of the advantages of this AIEgen over available molecular probes for the same purpose.

A report on incidence of COVID-19 among febrile patients attending a malaria clinic.

CONTEXT: Screening for malaria and coronavirus disease (COVID-19) in all patients with acute febrile illness is necessary in malaria-endemic areas to reduce malaria-related mortality and to prevent the transmission of COVID-19 by isolation. AIMS: A pilot study was undertaken to determine the incidence of SARS-CoV-2 infection among febrile patients attending a malaria clinic. SUBJECTS AND METHODS: All patients were tested for malaria parasite by examining thick and thin blood smears as well as by rapid malaria antigen tests. COVID-19 was detected by rapid antigen test and reverse transcriptase-polymerase chain reaction in patients agreeing to undergo the test. RESULTS: Out of 262 patients examined, 66 (25.19%) were positive for Plasmodium vivax, 45 (17.17%) for Plasmodium falciparum (Pf) with a slide positivity rate of 42.40%, and Pf% of 40.50%. Only 29 patients consented for COVID-19 testing along with malaria; of them, 3 (10.34%) were positive for COVID-19 alone and 2 (6.89%) were positive for both COVID-19 and P. vivax with an incidence of 17.24%. A maximum number of patients (196) did not examine for COVID-19 as they did not agree to do the test. CONCLUSION: Diagnosis of COVID-19 among three patients (10.34%) is significant both in terms of identification of cases and to isolate them for preventing transmission in the community. Detection of COVID-19 along with malaria is equally important for their proper management.

Dramatic Changes in Oligomerization Property Caused by Single Residue Deletion in Staphylococcus aureus Enolase.

Studies were conducted to understand the role of C-terminal lysine residues in the catalytic activity, structural stability and oligomeric properties of Staphylococcus aureus enolase. Interestingly, the S. aureus enolase, in solution, shows its presence as a stable dimer as well as the catalytically active fragile octamer. Compared to the hexa-histidine tagged S. aureus enolase (rSaeno), the deletion mutant showed the negligible difference in Km, but approximately 20-25% reduction in maximum reaction velocity (Vmax) and 2% reduction in turnover number were observed. These kinetic parameters indicate that K-434Δ deletion mutation does not drastically compromise the enzyme efficiency. The secondary structure and the octameric conformation of both the rSaeno and the K-434Δ mutant are very much stable between pH ranging from 6 to 9, temperatures ranging from 20 to 40 °C and in the presence of divalent metal ions Mg2+, Zn2+ and Mn2+. Under these conditions, the recombinant enzyme and the mutant are also catalytically very active. Intrinsic tryptophan fluorescence (320-380 nm) and CD spectral (195-260 nm) analysis revealed that the secondary structure and the surface architecture of the proteins are not majorly altered by the mutation. But, a significant correlation was observed between the time-dependent decrease in the catalytic activity and the oligomeric stability of rSaeno and K-434Δ mutant. The C-terminal lysine residues in the inter-dimer groove aid in folding and oligomerization of S. aureus enolase.

An overview of moonlighting proteins in Staphylococcus aureus infection.

Staphylococcus aureus is responsible for numerous instances of superficial, toxin-mediated, and invasive infections. The emergence of methicillin-resistant (MRSA), as well as vancomycin-resistant (VRSA) strains of S. aureus, poses a massive threat to human health. The tenacity of S. aureus to acquire resistance against numerous antibiotics in a very short duration makes the effort towards developing new antibiotics almost futile. S. aureus owes its destructive pathogenicity to the plethora of virulent factors it produces among which a majority of them are moonlighting proteins. Moonlighting proteins are the multifunctional proteins in which a single protein, with different oligomeric conformations, perform multiple independent functions in different cell compartments. Peculiarly, proteins involved in key ancestral functions and metabolic pathways typically exhibit moonlighting functions. Pathogens mainly employ those proteins as virulent factors which exhibit high structural conservation towards their host counterparts. Consequentially, the host immune system counteracts these invading bacterial virulent factors with minimal protective action. Additionally, many moonlighting proteins also play multiple roles in various stages of pathogenicity while augmenting the virulence of the bacterium. This has necessitated elaborative studies to be conducted on moonlighting proteins of S. aureus that can serve as drug targets. This review is a small effort towards understanding the role of various moonlighting proteins in the pathogenicity of S. aureus.

Studies on the gene regulation involved in the lytic-lysogenic switch in Staphylococcus aureus temperate bacteriophage Phi11.

Antirepressor proteins of bacteriophages are chiefly involved in interfering with the function of the repressor protein and forcing the bacteriophage to adopt the lytic cycle. The genome of Staphylococcus aureus phage, Phi11 has already been sequenced; from the genome sequence, we amplified gp07 gene and analysed its involvement in the developmental pathway of Phi11. Our results indicate that Gp07 functions as a novel antirepressor and regulates the developmental pathway of Phi11 by enhancing the binding of the Cro repressor protein to its cognate operator. We also report our finding that the CI repressor protein of Phi11 binds to the putative operator of Gp07 and regulates its expression. We further report that S.aureus transcriptional repressor LexA and coprotease RecA play a crucial role in the lytic-lysogenic switching in Phi11. We also identified that the N-terminal domain (Bro-N) of Gp07 is actually responsible for enhancing the binding of Cro repressor to its cognate operator. Our results suggest that Phi11 prophage induction is different from other bacteriophages. This study furnishes a first-hand report regarding the regulation involved in the developmental pathway of Phi11.

An Overview of Coronavirus COVID-19 with their Pathogenesis and Risk Assessment of the Disease Utilizing Positive Predictive Value of the Clinical and Laboratory Data.

COVID-19 has created a devastating pandemic, infecting more than 200 countries in its wake, only sparing Antarctica. The virus dissociates ferrous ion from the porphyrin ring of heme of haemoglobin-thus hampering the oxygen and carbon dioxide exchange in the lung and tissue. The toxic effect of ferrous (Fe2+) ions and carbon dioxide causes lung damage giving rise to severe respiratory distress and an often observed clotting disorder. Serum ferritin level is increased along with the rise of serum LDH, d-dimer, serum IL-6 and cardiac troponin. Associated leukocytosis, occasional lymphocytopenia and radiological changes of the lung are the pathological hallmarks of the disease. All these parameters including other clinical data such as age, fever, gender and associated co-morbidities may be used as a Risk Assessment tool for COVID-19 before the report of real-time polymerase chain reaction (RT PCR) is available. A timely intervention can contribute to rescuing millions from an untimely death.

Effect of ions and inhibitors on the catalytic activity and structural stability of S. aureus enolase.

The glycolytic enzyme enolase of Staphylococcus aureus is a highly conserved enzyme which binds to human plasminogen thereby aiding the infection process. The cloning, over expression and purification of S. aureus enolase as well as the effect of various metals upon the catalytic activity and structural stability of the enzyme have been reported. The recombinant enzyme (rSaeno) has been purified to homogeneity in abundant amounts (60 mg/L of culture) and the kinetic parameters (Km = 0.23 +/- 0.013 x 10-3 M; Vmax = 90.98 +/- 0.00052 U/mg) and the optimum pH were calculated. This communication further reports that increasing concentrations of Na+ ions inhibit the enzyme while increasing concentrations of K+ ions were stimulatory. In case of divalent cations, it was found that Mg2+ stimulates the activity of rSaeno while the rest of the divalent cations (Zn2+, Mn2+, Fe2+, Cu2+, Ni2+ and Ca2+) lead to a dose-dependent loss in the activity with a total loss of activity in the presence of Hg2+ and Cr2+. The circular dichroism data indicate that other than Hg2+, Ni2+ and to a certain extent Cu2+, none of the other ions destabilized rSaeno. The inhibitory roles of fluorides, as well as neurotoxic compounds upon the catalytic activity of rSaeno, have also been studied. Conformational changes in rSaeno (induced by ions) were studied using partial trypsin digestion.

Cloning, overexpression and purification of a novel two-domain protein of Staphylococcus aureus phage Phi11.

The genome of aureophage Phi11 reveals the presence of the gene gp07 which codes for the putative antirepressor protein (GenBank accession no. NC_004615.1). Antirepressor proteins are mainly involved in lytic cycle determination mechanisms of various bacteriophages. The Phi11 protein Gp07 consists of two domains-an amino terminal Bro domain and a carboxy terminal KilA domain. Despite the important role of antirepressor proteins in the developmental pathway of phages, there are no reports on the purification and characterization of aureophage antirepressor proteins. Here we describe a method to clone, overexpress and purify the full length Gp07 as carboxy terminal hexa histidine tag variant. The recombinant protein was expressed in Escherichia coli BL21(λDE3) cells and gradient of imidazole and NaCl were used for successful purification of the soluble recombinant protein to homogeneity. The protein exists as a dimer in solution as is evident from our gel filtration chromatography and glutaraldehyde cross-linking data. Further, we found that temperature has huge impact on the structural conformation of the protein. We expect that the purification of Gp07 will further our work in characterizing the role played by this protein during phage induction.

Expression of Phi11 Gp07 Causes Filamentation in Escherichia coli.

BACKGROUND: The Gp07 protein of aureophage Phi11 exhibits growth inhibitory effects when overexpressed in E. coli .The protein harbors two domains- an amino terminal Bro-like domain and a carboxy terminal Ant superfamily like KilA domain, of which the KilA domain retains the growth inhibitory effect of Gp07. METHODS: We studied the effects exerted by the overexpression of Gp07 and its separate domains upon the growth rate as well as the morphology of the E. coli cells. Additionally, we generated a mutant of Gp07 (designated as ΔGp07) by deleting the first eleven amino acid residues from the amino-terminal region of Gp07, and studied its growth inhibitory effects upon E. coli. RESULTS: Our results indicate that Gp07, ΔGp07 as well as the Carboxy-terminal region of Gp07 upon overexpression, retards the growth rate of the E. coli cells and also induces filamentation in the cells. Surprisingly, our data clearly suggests that the growth inhibition and filamentation induced by the the amino-terminal domain of Gp07 is temporal in nature. CONCLUSION: The carboxy-terminal of domain of gp07 is essential for its activity.

Changes in the Functional Activity of Phi11 Cro Protein is Mediated by Various Ions.

Phi11, a temperate bacteriophage of Staphylococcus aureus, has been found to harbor a cro repressor gene which facilitates Phi11 to adopt the lytic mode of development. The Cro protein has been found to bind very specifically to a 15-bp operator DNA, located in the Phi11 cI-cro intergenic region [1]. To investigate the effects exerted by different ions upon the interaction between Cro and its cognate operator DNA, we have employed gel shift assays as well as circular dichroism spectral analysis. In this communication, we have shown that NH4+ and acetate- ions better facilitated the binding of Cro with its cognate operator as compared to Na+, K+ and Li+. Interestingly, Mg2+, carbonate2- and Citrate3- have an inhibitory effect upon the binding. The effect of the said ions upon the structure of Cro was also investigated by circular dichroism and it was found that other than Citrate3- ions, none of the other ions destabilised the protein. On the other hand, Mg2+ and carbonate2- ions maintained the structure of the protein but severely hampered its functional activity. Citrate3- ions severely unfolded Cro and also inhibited its function. Considering all the data, NH4+ and acetate- ions appeared to be more suitable in maintaining the biological activity of Cro.