Exploring the evolutionary and pathogenic role of Acinetobacter baumannii biofilm-associated protein (Bap) through in silico structural modeling.

Acinetobacter species encode for extracellularly secreted Biofilm-associated protein (Bap), a multi-domain protein with variable molecular weights reaching several hundred kilodaltons. Bap is crucial for the development of multi-dimensional structures of mature biofilms. In our investigation, we analyzed 7338 sequences of A. baumannii from the NCBI database and found that Bap or Bap-like protein (BLP) was present in 6422 (87.52%) isolates. Further classification revealed that 12.12% carried Type-1 Bap, 68.44% had Type-2, 6.91% had Type-3, 0.05% had Type-6 or SDF-Type, and 12.51% lacked Bap or BLP. The majority of isolates with Type-1, Type-2, and Type-3 Bap belonged to ST1, ST2, and ST25, respectively. Phylogenetic analysis suggested that Type-1 Bap is the most ancient, while Type-3 and SDF-Type have evolved recently. Studying the interaction of predicted Bap structures with human CEACAM-1 and PIgR showed that Bap with its BIg13 and BIg6 domains interact with the N-terminal domain of CEACAM-1, involving Arg43 and Glu40, involved in CEACAM-1 dimerization. Also, we found that recently evolved Type-3 and SDF-Type Bap showed greater interaction with CEACAM-1 and PIgR. It can be asserted that the evolution of Bap has conferred enhanced virulence characteristics to A. baumannii with increased interaction with CEACAM-1 and PIgR. Using in silico approaches, this study explores the evolutionary, physicochemical, and structural features of A. baumannii Bap and unravels its crucial role in mediating interaction with human CEACAM-1 and PIgR through detailed structure modelling. These findings advance our understanding of A. baumannii Bap and highlight its role in pathogenesis.

Antibacterial and Antibiofilm Properties of the Alexidine Dihydrochloride (MMV396785) against Acinetobacter baumannii.

Antibiotic-resistant Acinetobacter baumannii infections among patients in hospital settings are rising at an alarming rate. The World Health Organization has designated carbapenem-resistant A. baumannii as a priority pathogen for drug discovery. Based on the open drug discovery approach, we screened 400 compounds provided as a Pandemic Response Box by MMV and DNDi to identify compounds with antibacterial and antibiofilm activity against two A. baumannii reference strains using a highly robust resazurin assay. In vitro screening identified thirty compounds with MIC ≤ 50µM having growth inhibitory properties against the planktonic state. Five compounds, with MMV IDs MMV396785, MMV1578568, MMV1578574, MMV1578564, and MMV1579850, were able to reduce metabolically active cells in the biofilm state. Of these five compounds, MMV396785 showed potential antibacterial and antibiofilm activity with MIC, MBIC, and MBEC of 3.125 µM, 12.5, and 25-100 µM against tested A. baumannii strains, respectively, showing biofilm formation inhibition by 93% and eradication of pre-formed biofilms by 60-77.4%. In addition, MMV396785 showed a drastic reduction in the surface area and thickness of biofilms. Further investigations at the molecular level by qRT-PCR revealed the downregulation of biofilm-associated genes when exposed to 50 µM MMV396785 in all tested strains. This study identified the novel compound MMV396785 as showing potential in vitro antibacterial and antibiofilm efficacy against A. baumannii.

Factors mediating Acinetobacter baumannii biofilm formation: Opportunities for developing therapeutics.

Acinetobacter baumannii has notably become a superbug due to its mounting risk of infection and escalating rates of antimicrobial resistance, including colistin, the last-resort antibiotic. Its propensity to form biofilm on biotic and abiotic surfaces has contributed to the majority of nosocomial infections. Bacterial cells in biofilms are resistant to antibiotics and host immune response, and pose challenges in treatment. Therefore current scenario urgently requires the development of novel therapeutic strategies for successful treatment outcomes. This article provides a holistic understanding of sequential events and regulatory mechanisms directing A. baumannii biofilm formation. Understanding the key factors functioning and regulating the biofilm machinery of A. baumannii will provide us insight to develop novel approaches to combat A. baumannii infections. Further, the review article deliberates promising strategies for the prevention of biofilm formation on medically relevant substances and potential therapeutic strategies for the eradication of preformed biofilms which can help tackle biofilm-associated A. baumannii infections. Advances in emerging therapeutic opportunities such as phage therapy, nanoparticle therapy and photodynamic therapy are also discussed to comprehend the current scenario and future outlook for the development of successful treatment against biofilm-associated A. baumannii infections.

Development of Two-Tube Loop-Mediated Isothermal Amplification Assay for Differential Diagnosis of Plasmodium falciparum and Plasmodium vivax and Its Comparison with Loopamp™ Malaria.

To strengthen malaria surveillance, field-appropriate diagnostics requiring limited technical resources are of critical significance. Loop-mediated isothermal amplification (LAMP) based malaria diagnostic assays are potential point-of-care tests with high sensitivity and specificity and have been used in low-resource settings. Plasmodium vivax-specific consensus repeat sequence (CRS)-based and Plasmodium falciparum-specific 18S rRNA primers were designed, and a two-tube LAMP assay was developed. The diagnostic performance of a closed-tube LAMP assay and Loopamp™ Malaria Detection (Pan/Pf, Pv) kit was investigated using nested PCR confirmed mono- and co-infections of P. vivax and P. falciparum positive (n = 149) and negative (n = 67) samples. The closed-tube Pv LAMP assay showed positive amplification in 40 min (limit of detection, LOD 0.7 parasites/µL) and Pf LAMP assay in 30 min (LOD 2 parasites/µL). Pv LAMP and Pf LAMP demonstrated a sensitivity and specificity of 100% (95% CI, 95.96-100% and 89.85-100%, respectively). The LoopampTM Pan/Pf Malaria Detection kit demonstrated a sensitivity and specificity of 100%, whereas LoopampTM Pv showed a sensitivity of 98.36% (95% CI, 91.28-99.71%) and specificity of 100% (95% CI, 87.54-100%). The developed two-tube LAMP assay is highly sensitive (LOD ≤ 2 parasite/µL), demonstrating comparable results with the commercial Loopamp™ Malaria Detection (Pf/pan) kit, and was superior in detecting the P. vivax co-infection that remained undetected by the Loopamp™ Pv kit. The developed indigenous two-tube Pf/Pv malaria detection can reliably be used for mass screening in resource-limited areas endemic for both P. falciparum and P. vivax malaria.

Colistin Resistance Among Multiple Sequence Types of Klebsiella pneumoniae Is Associated With Diverse Resistance Mechanisms: A Report From India.

Background: The resistance to colistin and carbapenems in Klebsiella pneumoniae infections have been associated with increased morbidity and mortality worldwide. A retrospective observational study was conducted to determine the prevalence and molecular events contributing to colistin resistance. Methods: Clinical samples were screened for colistin resistance and underlying mechanisms were studied by PCR-based amplification and sequence analysis of genes of two-component regulatory system (phoPQ and pmrAB), regulatory transmembrane protein-coding mgrB, and mobilized colistin resistance genes (mcr-1-8). Gene expression of pmrC and pmrK was analyzed by qRT-PCR, and the genetic relationship was assessed by MLST. The putative effect of amino-acid substitutions was predicted by a combination of bioinformatics tools. Results: Of 335 Klebsiella spp. screened, 11 (3.2%) were identified as colistin-resistant (MIC range, 8 to >128 µg/ml). K. pneumoniae isolates belonged to clonal complex-11 (CC11) with sequence types (STs): 14, 16, 43, 54, 147 and 395, whereby four isolates conferred three novel STs (3986, 3987 and 3988) profiles. Sequence analysis revealed non-synonymous potentially deleterious mutations in phoP (T151A), phoQ (del87-90, del263-264, L30Q, and A351D), pmrA (G53S), pmrB (D150V, T157P, L237R, G250C, A252G, R315P, and Q331H), and mgrB (C28G) genes. The mgrB gene in three strains was disrupted by insertion sequences encoding IS1-like and IS5/IS1182 family-like transposase genes. All 11 isolates showed an elevation in the transcription level of pmrC gene. Mobilized colistin-resistance (mcr) genes were not detected. All but one of the colistin-resistant isolates was also resistant to carbapenems; ß-lactamase genes blaNDM-1-like , blaOXA-48-like , and blaCTX-M-like were detected in eight, five, and nine isolates, respectively. Conclusion: All the studied colistin- and carbapenem-resistant K. pneumoniae isolates were genetically distinct, and various mechanisms of colistin resistance were detected, indicating its spontaneous emergence in this bacterial species.

Allelic variation of msp-3α gene in Plasmodium vivax isolates and its correlation with the severity of disease in vivax malaria.

Malaria is a global socio-economic burden of which Plasmodium vivax contributes for about 70-80 million cases on an annual basis worldwide and 60-65% cases in India. Diversity observed in highly polymorphic Merozoite Surface Protein-3α (msp-3α) encoded by MSP-3 gene family, has been used efficiently for genotyping of P. vivax infection. This study aims to correlate the severity of clinical symptoms with parasite load, genotype of P. vivax and multiplicity of infection. Based on clinical symptoms classification, 31 (67.9%) out of 46 cases were found to be severe while 15 (32.6%) were non-severe and correlation of the severity of vivax infection with parasite load was not observed. Analysis of msp3-α allele genotype showed that out of 31 severe cases, 19 (61.2%) were single-clone infection cases whereas 12 (38.7%) were multi-clone infections. Similarly, out of 15 non-severe cases, 9 (60%) were single clone and 6 (40%) were multi-clone infections indicating the absence of a correlation between the multiplicity of infection and disease severity. Allele frequency observed was 65.9%, 23.4%, 23.4%, and 28.2% for allele A, B, C and D, respectively. An important finding was the greater distribution of allele D than alleles B and C, which has been reported as a rare allele otherwise. Further, of 13 cases with allele D, 76.9% (10/13) cases were severe. This study showed the absence of a correlation between the severity of clinical symptoms with parasite load and multiplicity of infection but at the same time drives a possibility of severe vivax malarial symptoms to have an association with the persistence of allele D in the population. This upon exploration can lead to the development of a target in detection of severe cases of malaria.