Hospital-acquired infections (HAIs) are rapidly increasing on a global scale, driven by factors such as inadequate facility maintenance, ineffective infection control practices, resistance development in pathogens, increasing cross and co-infections, etc. The magnitude of this problem is substantial and warrants immediate attention. Our study targets the nosocomial lower respiratory tract bacterial pathogens which are frequently reported to cause severe infections. K. pneumoniae is a common opportunistic pathogen that causes nosocomial infections, particularly in immunocompromised patients, leading to fatal disease manifestations such as meningitis, chronic pneumonia, necrotizing fasciitis, etc.^25,26 S. pneumoniae-induced pneumococcal community-acquired pneumonia (pCAP) is a major cause of LRTIs in the elderly, often leading to disorientation, confusion, and exacerbation of pre-existing conditions; and morbidity and mortality among children. Although, S. pyogenes rarely causes LRTIs, it can lead to severe and fatal pneumonias when infects. S. aureus is the most common Gram-positive bacterium in health care systems. Therefore, the present study was focused around four key bacterial pathogens, viz. S. pyogenes, S. aureus, S. pneumoniae, and K. pneumoniae, which are commonly implicated in nosocomial lower respiratory tract infections.²⁷

A major risk factor associated with these pathogens is, the rise in MDRs which undermines various strategies aimed at mitigating nosocomial infections.^28,29 For instance, S. aureus, shows a resistance rate of nearly 83% to Penicillin G. Of the several reasons attributed to the inefficacy or resistance seen in commercially used beta-lactam antibiotics, a suboptimal binding affinity (between -6 to -9 kcal/mol) along with a small binding pocket size has been amongst the most prevalent. Beta-lactam antibiotics, such as penicillin, target PBPs, which are required for bacterial cell wall formation. However, when the binding affinity of these antibiotics falls between -6 and -9 kcal/mol, the interaction may be insufficient to effectively block the PBPs, resulting in diminished effectiveness. Furthermore, the size restrictions of the PBP binding pocket can prohibit bigger or structurally modified antibiotics from binding well, lowering the drug’s efficacy.³⁰ Among macrolides, erythromycin has been documented with the highest resistance rate (51.9%) since 2019, with a notable increase in levofloxacin resistance, ranging from 28.1% to 54.9%. Additionally, K. pneumoniae, a Gram-negative bacterium demonstrated an average resistance rate of 70% to cephalosporin and penicillin. The increasing prevalence of MDR pathogens accentuates necessity for the discovery of novel antimicrobial compounds with improved antimicrobial structural capabilities.²⁸

Actinomycetes have demonstrated as a powerful source of antibiotics in the last few decades. However, much of the Actinomycetes studied have been soil-borne, the marine counterparts are underexplored. Actinomycetes found in marine environments are abundant in bioactive compounds exhibiting unique properties. Unlike their terrestrial kin, marine Actinomycetes have developed unique metabolic pathways to adapt to the marine environment. This in turn leads to the production of many unique compounds with distinct structures and functions. These microorganisms thrive in diverse marine habitats, including sediments, seawater, and marine invertebrates, contributing significantly to the biodiversity of marine ecosystems. Marine Actinomycetes derived compounds are highly used in biotechnology and drug discovery.^31-33 Many novel promising bioactive metabolites such as, streptopyrrolidine, cyclo-(l-Pro-l-Met), and thiocoraline were discovered from marine Actinomycetes in a recent study.^7,34,35 In this regard, our study successfully isolated many marine Actinomycetes and also identified a potential Actinomycete strain, S. rochei SZ33. This strain, originating from a relatively underexplored marine habitat, demonstrated significant antimicrobial activity, effectively inhibiting the target pathogens by the production of antimicrobial metabolites. Utilising S. rochei SZ33 as a source of bioactive compounds warrants further investigation for exploring all possible therapeutic applications. A recent study highlighted the antimicrobial activity of Streptomyces rochei SUN35, against MRSA, Fusarium solani, Aspergillus fumigatus, Pseudomonas aeruginosa, and Penicillium chrysogenum under in vitro conditions.³⁶ This emphasizes the potential of S. rochei species in combating a wide range of pathogenic microorganisms.

To improve the production of the secondary metabolites, and the consequential heightened inhibition of target pathogens, media composition and physical conditions were optimized.³⁷ Optimization studies indicated that the highest production of antimicrobial secondary metabolites occurred at 25 °C and pH 6, with a medium containing starch as the carbon source, yeast extract as the nitrogen source, and a salt concentration of 2%. These optimized conditions are easily achievable, and beneficial for the large-scale production of antimicrobials employing S. rochei SZ33. Post optimization, the inhibition was increased by two-fold against the tested pathogens. Metabolite analysis using GC-MS identified several bioactive compounds,³⁸ including β-carotene-3,3′-diol, (3R,3’R)-all-trans, 2H-pyran,³⁹ 2-(7-heptadecynyloxy) tetrahydro, and astaxanthin,^4,17 all of which demonstrated antimicrobial properties, while aurin compounds were identified as LsrK inhibitors.⁴⁰ The observed inhibition of the target pathogens was significantly higher than that of the standard antibiotic, might well be a consequence of inherent synergism present between the metabolites of the crude extract.

The antimicrobial activity of astaxanthin-alpha tocopherol has been shown to be due to its ability to disrupt bacterial cell membranes.⁴¹ Previous studies have demonstrated that secondary metabolites produced by marine Actinomycetes exhibit superior antimicrobial activity against respiratory pathogens, including S. pneumoniae, Mycobacterium tuberculosis, K. pneumoniae, and S. aureus, which are commonly associated with pneumonia and other respiratory infections. Marine ecosystems are generally isolated and diversified, with harsh circumstances such as high salinity, fluctuating temperatures, and tremendous pressure. To survive this, the said microorganisms have developed the ability to synthesize new chemical substances. The chemical diversity of secondary metabolites in marine Actinomycetes is primarily driven by the evolutionary pressure to compete with other microorganisms in the competitive environment. This explains why metabolites derived from marine actinomycetes possess distinct inhibitory modes of action, circumventing the resistance mechanisms that many infections have acquired against standard antibiotics.³¹

Several bioactive compounds derived from marine Actinomycetes have shown significant potential as therapeutic agents for treating respiratory infections. For instance, Streptomyces sp. 6921, isolated from marine sediments in Mauritius, secretes a range of antibacterial compounds, including C-glycosides himalomycins, anthraquinones, fridamycin E, and chromophore.⁴² Additionally, a marine Streptomyces sp. 182SMLY was shown to produce streptophenazines, which inhibited (MRSA). Another strain, S. psammoticus, produced a novel polyketide tetracycline analog, SBR-22, which also demonstrated potent activity against MRSA.⁴³

The proliferation of multidrug-resistant organisms is severely undermining the treatment using existing drugs.^44,45 This accentuates the urgent need for the development of novel drugs and therapeutic strategies that are effective against antibiotic-resistant pathogens.^46-49 Crude extracts, which contain a diverse array of active metabolites, offer a promising alternative to traditional single or combination drug therapies, as their complex composition may provide enhanced efficacy through synergistic interactions among the bioactive compounds.

The compounds might be new antimicrobial agents that mitigate antibiotic resistance. Several of these compounds bind strongly to bacterial enzymes, disrupting processes such as cell wall synthesis, DNA replication, and enzyme activity. In addition to hydrophobic interactions, hydrogen bonds, and a double-stacked structure, these molecules are able to bind to microbial targets with exceptional stability. By addressing both traditional pathways and new strategies to combat antibiotic-resistant pathogens, this study reveals a promising mechanism of action. Integrating these molecular insights with marine actinomycetes underscores their potential as a source of innovative antimicrobial therapies.

In this regard, the compounds identified by GC-MS were further analyzed by docking them with PBPs as molecular targets for all the pathogens associated with nosocomial LRTIs. PBPs, the essential enzymes in bacterial cell wall synthesis, are targeted by antibiotics such as Penicillin and Carbapenems, which inhibit their function and lead to bacterial cell death. However, the extensive use of these antibiotics has driven the evolution of drug resistance and multidrug-resistant pathogens, reducing the effectiveness of these drugs. This in turn has led to PBPs being frequently employed in docking studies for nosocomial (hospital-acquired) infections as it is the key target of beta-lactam antibiotics. PBPs being elemental for bacterial cell wall formation, and their blockage causes cell death. Nosocomial bacteria predominantly develop resistance to beta-lactam antibiotics by altering PBPs, thereby reducing the antibiotic’s affinity for these enzymes. This makes PBPs an ideal target for research into finding new therapeutic targets.

This study provides compelling evidence for the potential of naturally derived compounds as potent inhibitors of PBPs across a range of pathogenic bacteria. The findings demonstrate that these natural compounds exhibit superior binding affinities compared to conventional synthetic antibiotics. The GC-MS analysis identified 28 distinct compounds in the crude extract, many of which displayed notable antimicrobial capabilities. Compounds such as zeaxanthin, lutein, and astaxanthin exhibited exceptionally strong interactions with PBPs, compared to commercial antibiotics, suggesting their promise as alternatives or adjuncts in combating antibiotic resistance.

Zeaxanthin and lutein demonstrated strong binding affinities to the PBP3 enzyme of S. aureus, with binding energies of -9.9 kcal/mol and -9.7 kcal/mol, respectively. The antibacterial mode of action, in carotenoids ranges from dissolution of the cell membrane, disruption of cell wall synthesis and alterations in the immune response. Their interactions with key amino acid residues, such as TYR275B and GLN548A, suggest a capacity for competitive inhibition, which could effectively mitigate resistance mechanisms in this pathogen. Furthermore, these carotenoids also exhibited robust binding to the PBP3 enzymes of P. aeruginosa and K. pneumoniae, with binding affinities of -9.5 kcal/mol and -9.3 kcal/mol, respectively. The antimicrobial actions of these compounds include enhancing oxidative stress in bacteria and inhibiting biofilm formation.

Astaxanthin, another naturally derived compound, exhibited a binding affinity of -9.4 kcal/mol for the PBP2 enzyme of P. aeruginosa, suggesting its potential as an alternative to antibiotics like doripenem. Astaxanthin is known for its potent antioxidant properties, which can reduce inflammation and enhance the immune response against bacterial infections. Furthermore, this compound has the capacity to disrupt bacterial cell membranes, subsequently resulting in cell death. The analysis also revealed that the carotenoids lutein and zeaxanthin maintained significant binding affinities of -8.3 kcal/mol and -8.1 kcal/mol, respectively, against the PBP targets of S. pneumoniae. These naturally occurring compounds have demonstrated the ability to inhibit cell wall synthesis and interfere with bacterial metabolic pathways, offering an effective alternative strategy to control S. pneumoniae infections compared to traditional antibiotics like amoxicillin. In addition to substantial differences in binding affinities from commercial antibiotics against PBPs, the naturally derived compounds identified by GC-MS also displayed an alternate binding pocket along with a significant difference in binding pocket sizes. Larger or more open binding pockets may allow for easier entry of larger molecules or those with bulky groups. If the size of the binding pocket is too small, steric hindrance can prevent effective binding of larger compounds, reducing their antibacterial activity. A bigger pocket may also provide more structural flexibility, allowing for the binding of a wider spectrum of substrates or inhibitors. Larger pockets frequently offer more possibilities for interaction between the antimicrobial drug and the target protein. Binding of antimicrobial agents to alternate sites other than the active sites could lead to inhibition of the enzyme without changing the active site.

In addition to the carotenoids, zeaxanthin, lutein, and astaxanthin, several other natural compounds, including carbamic acid derivatives, rhodopin, phenyl-beta-D-glucoside, and aurin, have demonstrated the potential to inhibit synthesis of cell wall by binding to PBPs across various pathogenic strains, as evidenced by molecular docking analyses. Furthermore, certain other naturally derived compounds, such as fatty acid methyl esters and polyunsaturated fatty acids, may exert antimicrobial effects through alternative mechanisms, which may involve increasing membrane fluidity and permeability, promoting lipid peroxidation, or disrupting membrane integrity, ultimately leading to bacterial cell death. There is limited direct evidence for antibacterial activity from compounds such as lutein, zeaxanthin, astaxanthin, etc. However, studies have looked at their ability to work synergistically either with other similar compounds or with synthetic antibiotics.⁴²

The results from the study perhaps favor the argument of using crude extracts as superior antimicrobials. Combining chemicals with varied binding affinities, and allosteric binding pockets could improve antibacterial activity, particularly against resistant species. This method reduces the possibility of resistance building.⁴³