Prakash Narayana Reddy

Staphylococcus aureus ( S. aureus ) is a major human pathogen that causes a wide range of superficial skin infections to the life-threatening systemic diseases. Emergence and increasing prevalence of anti-biotic resistance S. aureus strains have accelerated the need for effective vaccines. As S. aureus pathogenesis involve its surface antigens to facilitate the colonization and invasion into host, targeting these surface antigens represent a promising strategy for S. aureus vaccine development. In this study, we evaluated the efficacy of two surface antigens of S. aureus -fibronectin-binding protein A (FnBPA) and lipoteichoic acid (LTA) as vaccine candidates. We expressed the N-terminal region of FnBPA in Escherichia coli and mixed with LTA to generate r-FnBPA N _1-3 +LTA antigen mixture, emulsified with Freund’s adjuvant as r-FnBPA N _1-3 +LTA antigen mixture or r-FR alone and immunized to BALB/c mice. Mice immunized with r-FnBPA N _1-3 +LTA mixture displayed a strong antibody response, strong bacterial growth inhibition in vitro and increased survival following lethal S. aureus challenge compared with mice immunized with r-FnBPA N _1-3 alone. These findings suggest that incorporation of the S. aureus surface antigens like FnBPA with LTA provides improved protective efficacy and highlights the potential of a multi-antigen vaccine strategy for preventing S. aureus infections.

Enterobacteriaceae pathogens like E. coli, Shigella and Salmonella are mainly associated with the food poisoning and various mucosa associated infections. These pathogens invade the host and rely on the host derived nutrients for their survival. One such nutrient is sialic acid (N-acetylneuraminic acid is the common one) which is present in the mucosal linings throughout the body. Ability of these pathogens to breakdown and utilize sialic acid for their growth is a crucial factor for their survival and pathogenesis. However, the heterogeneity among these enteric pathogens remains unstudied. In this study, we explored the difference in the sialic acid utilization pattern among these pathogens and found that the interactions between the N-acetylneuraminic acid (Neu-5-Ac) and the NanA, an enzyme that catalyze the breakdown of sialic acid play a key role for this heterogeneity. Additionally, we found that the Neu-5-Ac interacted differently with the NanA from each strain because of the variations in the active sites/contact sites which made the strains to utilize sialic acid different. Finally, our results provide an insight into the current understanding of sialiobiology research by highlighting the active site/contact sites differences drived the pathogens’ ability to adapt to diverse host environments, potentially altering their pathogenicity and resistance to host immune responses. This study provides new insights into the evolution of Enterobacteriaceae and suggest potential targets for therapeutic intervention aimed at disrupting sialic acid utilization.

Enterobacteriaceae pathogens such as Escherichia coli, Salmonella sp., Shigella sp., Proteus sp., and Klebsiella pneumoniae cause a wide range of gastrointestinal and other mucosal infections. These bacteria acquire antibiotic resistance very quickly and evolve into multi-drug resistant strains thereby making the treatment very difficult. The outer membrane proteins (OMPs) in Enterobacteriaceae are potential vaccine candidates owing for their high immunogenicity and amino acid conservation. The OmpA is one such protein which need to be investigated for the development of a potential subunit vaccine against multiple infections casued by the pathogens of Enterobacteriaceae. To investigate this, we expressed and purified the highly conserved OmpA of S. typhimurium and studied the antibody mediated cross reactivity with the other Enterobacteriaceae pathogens. This was validated through dot ELISA performed with the hyperimmune sera raised against rOmpA of S. typhimurium. We further analyzed the sequence of OmpA protein and clearly understood that the B-cell epitopes in the protein are highly conserved are responsible for cross reactivity among the Enterobacteriaceae pathogens. This work led to findings that provide strong evidence for the application of OmpA in broad-spectrum subunit vaccine against enteric infections.

Klebsiella pneumoniae is one of the major nosocomial pathogens responsible for pneumoniae, septicaemia, liver abscesses, and urinary tract infections. Coordinated efforts by antibiotic stewardship and clinicians are underway to curtail the emergence of antibiotic-resistant strains. The objective of the present study is to characterize K. pneumoniae strains through antibiotic resistance screening for production of beta-lactamases (β-lactamases) such as extended spectrum beta lactamases (ESBLs), AmpC β-lactamases, and carbapenemases by phenotypic and genotypic methods and genetic fingerprinting by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and repetitive element palindromic PCR (REP-PCR). A total of 85 K. pneumoniae strains isolated from 504 human urinary tract infections (UTI) were used in this study. Only 76 isolates showed positive in phenotypic screening test (PST), while combination disc method (CDM) as phenotypic confirmatory test (PCT) confirmed 72 isolates as ESBL producers. One or more β-lactamase genes were detected by PCR in 66 isolates (91.66%, 66/72) with bla_TEM gene being the most predominant (75.75%, 50/66). AmpC genes could be detected in 21 isolates (31.8%, 21/66) with FOX gene being the predominant (24.24%, 16/66), whereas NDM-I was detected in a single strain (1.51%, 1/66). Genetic fingerprinting using ERIC-PCR and REP-PCR revealed wide heterogeneity among β-lactamase producing isolates with discriminatory power of 0.9995 and 1, respectively.

Staphylococcus aureus is a natural commensal microflora of humans which causes opportunistic infections due to its large arsenal of exotoxins, invasion, immune evasion, and antibiotic resistance mechanisms. The primary goal of this study is to develop a multiplex PCR (mPCR) assay for simultaneous detection of Staphylococcus aureus (nuc) and its virulence genes coding for prominent exotoxins namely alpha hemolysin (hla), enterotoxins A (sea), enterotoxin B (seb), toxic shock syndrome toxin (tsst-1), and the gene coding for methicillin resistance (mecA). A competitive internal amplification control (IAC) was included in the assay to exclude the false negative outcomes. Highly specific primer pairs were designed for the target genes using in silico resources. At the outset, monoplex PCRs were standardized using reference S. aureus strains. Primer specificity to the target genes was authenticated through restriction digestion analysis of amplified PCR products. Multiplex PCR was optimized in increments of one gene starting with nuc and IAC amplified simultaneously using one pair of primers (nuc) in a competitive manner. The mPCR assay was found to be highly sensitive with a detection limit of ~10 CFUs per reaction for pure cultures. Multiplex PCR assay was further evaluated on the retail and processed food samples to test the prevalence of S. aureus and study their exotoxin profiles. Of the 57 samples examined, 13 samples (22.80%) were found to be contaminated with S. aureus whose DNA was extracted after a 6-h enrichment period. Among these, a high percentage of hemolytic and enterotoxin A positive strains were encountered. The mPCR assay developed in this study would be a useful tool for rapid and reliable monitoring of S. aureus for food quality testing and from clinical infections.

Staphylococcus aureus is a serious pathogen unleashing its virulence through several classes of exotoxins such as hemolysins and enterotoxins. In this stud

In this study, 504 urine samples collected from patients suffering from UTIs were screened for presence of K. pneumoniae. The overall occurrence of Klebsiella spp. and K. pneumoniae was found to be 23.2% (117/504) and 16.8% (85/504) respectively. Antibiotic susceptibility testing of 85 K. pneumoniae isolates was carried out by disc diffusion which revealed alarming levels of antibiotic resistance. Antimicrobial resistance was prominently observed against cefpodoxime (76.47%) followed by ampicillin (70.59%), ceftriaxone (52.94%), cefoxitin (50.59%), amoxyclav (48.24%), ofloxacin (45.88%), cefotaxime (44.71%), cefepime (43.53%) and doxycycline hydrochloride (40%). A small percentage of strains also exhibited resistance to other antimicrobials in the range of 7 – 35 %. Around 77.6% of the isolates were found to be resistant to three or more antibiotic classes and 66.7% of the isolates had multiple antibiotic resistance (MAR) index values >0.2. Screening of virulence genes in 85 K. pneumoniae isolates revealed that uge gene was the most predominant (11/85, 12.9%), followed by rmpA (9/85, 10.5%), kfu (4/85, 4.7%) and aerobactin genes (2/85, 2.35%). Further, the overall percentage of biofilm producers were found to be 17.65% (15/85). This study warrants hospitals and health care centres to reduce misuse of antibiotics and manage UTI with appropriate treatment after performing antibiotic susceptibility testing.

In the present study, immunoglobulin Y (IgY) antibodies were raised in hens against the surface staphylococcal protein A (SpA) of Staphylococcus aureus. Anti-SpA IgY were tested in vitro for diagnostic applications, bacteriostatic, and biofilm inhibition effects. A specific and sensitive immunocapture PCR (IPCR) was developed to detect S. aureus from food, clinical, and environmental samples. Anti-SpA IgY were used for capturing S. aureus cells from different matrices. Chicken antibodies were chosen over mammalian antibodies based on its inertness to immunoglobulin (Ig)-binding property of SpA protein. No cross-reactivity was encountered with closely related Gram-positive and Gram-negative food pathogens. Inter-assay variation is < 10%. The assay was found suitable for testing on solid and liquid food samples, skin, and nasal swabs. The assay showed limit of detection of ≥ 10² CFU/mL from broth cultures and 10² to 10³ CFU/ml from diverse natural samples. This assay overcomes the false positives commonly encountered while using mammalian immunoglobulins (IgG). Anti-SpA IgY antibodies were tested for their bacteriostatic effect on the growth of S. aureus. IgY antibodies at a concentration of 150 μg/ml inhibited the growth of S. aureus completely indicating the potential of IgY antibodies in neutralization of infectious pathogens. Similarly, anti-SpA IgY at MIC50 concentration reduced biofilm formation by ~ 45%. In view of advantages offered by IgY antibodies for specific detection of S. aureus in immunocapture PCR (IPCR) assay and in vitro neutralization potential of S. aureus, we recommend using IgY over conventional IgG of mammals involving S. aureus and its antigens. Key points • IPCR with anti-SpA IgY for S. aureus was specific and sensitive for natural samples. • Anti-SpA IgY at 150 ug/ml displayed growth inhibition of S. aureus strains temporarily. • Anti-SpA IgY at MIC50 concentrations inhibited the biofilm formation partially.

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.