Infections due to antibiotic resistant bacteria are increasing globally and this needs immediate attention. Bacteriophages are considered an effective alternative for the treatment of bacterial infections. The aim of this study was to isolate and characterize the bacteriophages that infect Escherichia coli, Klebsiella pneumoniae, and Enterobacter species. For this, clinical bacterial isolates of the mentioned species were obtained from diagnostic centers located in Chennai, Tamil Nadu, India. The bacteriophages were isolated from sewage water samples collected from Tamil Nadu, India. Phage isolation was performed using enrichment method and agar overlay method was used to confirm the presence of bacteriophages. All the phages were characterized for their life cycle parameters, genome analysis, and in vitro phage cocktail activity. The three bacteriophages exhibited broad host range activity: Escherichia virus myPSH2311 infecting E. coli belonging to six different pathotypes, Klebsiella virus myPSH1235 infecting K. pneumoniae belonging to four different serotypes and Enterobacter virus myPSH1140 infecting four different species of Enterobacter. Morphological observations suggested that the bacteriophages belonged to, Phieco32virus (Escherichia virus myPSH2311), Podoviridae (Klebsiella virus myPSH1235), and Myoviridae (Enterobacter virus myPSH1140). The life cycles (adsorption, latent period, and cell burst) of Escherichia virus myPSH2311, Klebsiella virus myPSH1235 and Enterobacter virus myPSH1140 were found to be 26, 40, and 11 min, respectively. Genomic analysis revealed that Escherichia virus myPSH2311 is closely related to Escherichia phage vB_EcoP_SU10, Klebsiella virus myPSH1235 is closely related to Klebsiella phage vB_KpnP_KpV48 and Enterobacter virus myPSH1140 is closely related to Enterobacter phage PG7 and Enterobacter phage CC31. When phage cocktail was used against multiple bacterial mixtures, there was a reduction in bacterial load from 10⁶ to 10³ CFU/mL within 2 h. All the three characterized phages were found to have a broad host range activity and the prepared phage cocktails were effective against mixed bacterial population that are resistant to meropenem and colistin, two last resort antibiotics. Infections caused by drug resistant bacteria will be a serious threat in the future and the use of virulent bacteriophages in therapy may offer an effective solution.

Delivery of therapeutic compounds to the site of action is crucial. While many chemical substances such as beta-lactam antibiotics can reach therapeutic levels in most parts throughout the human body after administration, substances of higher molecular weight such as therapeutic proteins may not be able to reach the site of action (e.g., an infection) and are therefore ineffective.

Pulmonary diseases of viral origin are often followed by the manifestation of secondary infections, leading to further clinical complications and negative disease outcomes. Thus, research on secondary infections is essential. Here, we review clinical data of secondary bacterial infections developed after the onset of pulmonary viral infections. We review the most recent clinical data and current knowledge of secondary bacterial infections and their treatment in SARS-CoV-2 positive patients; case reports from SARS-CoV, MERS-CoV, SARS-CoV2 and the best-studied respiratory virus, influenza, are described. We outline treatments used or prophylactic measures employed for secondary bacterial infections. This evaluation includes recent clinical reports of pulmonary viral infections, including those by COVID-19, that reference secondary infections. Where data was provided for COVID-19 patients, a mortality rate of 15.2% due to secondary bacterial infections was observed for patients with pneumonia (41 of 268). Most clinicians treated patients with SARS-CoV-2 infections with prophylactic antibiotics (63.7%, n = 1,901), compared to 73.5% (n = 3,072) in all clinical reports of viral pneumonia included in this review. For all cases of viral pneumonia, a mortality rate of 10.9% due to secondary infections was observed (53 of 482). Most commonly, quinolones, cephalosporins and macrolides were administered, but also the glycopeptide vancomycin. Several bacterial pathogens appear to be prevalent as causative agents of secondary infections, including antibiotic-resistant strains of Staphylococcus aureus and Klebsiella pneumoniae.

Phage therapy is one of the promising alternatives to combat the increasing problem of antibiotic resistance. Lyophilization is used for the preparation of pharmaceutical products to improve their stability in long-term storage. The aim of this study was to improve the stability of lyophilized bacteriophages using different excipients. Three lytic bacteriophages Escherichia phage ECP311, Klebsiella phage KPP235 and Enterobacter phage ELP140 were subjected to lyophilization using six different excipients: glucose, sucrose, gelatin, mannitol, polyethylene glycol and sorbitol. The lyophilized phages were stored at 4 °C and 37 °C and rehydrated using biological saline to test their viability at 5 months interval up to 20 months. The results showed that the use of sucrose, gelatin and their combination was beneficial in maintaining the viability of phages post-lyophilization. When lyophilized phages were stored at 4 °C, their viability was maintained up to 20 months, but at 37 °C there was a reduction in activity after 10 months. This is one of the few studies to report the lyophilization of phage cocktails to have viability for up to 10 months. Our study identified promising lyophilization excipients to effectively lyophilize bacteriophages for pharmaceutical applications and long-term storage.

Phage therapy is the use of lytic bacteriophages to cure infections caused by bacteria. The aim of this study is to isolate and to characterize the bacteriophages against Escherichia coli isolated from clinical samples. For isolation of bacteriophages, water samples were collected from the Ganges River, and phage enrichment method was followed for phage isolation. Microbiological, genomic and lyophilization experiments were carried out to characterize the bacteriophage. Galleria mellonella was used to study the potential of phages against E. coli infection. Escherichia phage myPSH1131 belonging to Podoviridae family and found to have broad host range infectivity (n = 31) to infect Enterohemorrhagic E. coli (n = 9), Enteropathogenic E. coli (n = 6), Enterotoxigenic E. coli (n = 3), Enteroaggregative E. coli (n = 3), Uropathogenic E. coli (n = 9) and one unknown E. coli. The genome size is 76,163 base pairs (97 coding regions) and their genes show high similarity to SU10 phage. Lyophilization studies showed that the use of 1M sucrose, 2% gelatin and the combination of both 0.5M sucrose plus 1% gelatin could restore phage viability up to 20 months at 4°C. For in vivo studies, it was observed that a single phage dose can reduce the E. coli infection but to achieve 100% survival rate the infected larvae should be treated with three phage doses (20 μL, 10³ PFU/mL) at 6 hours interval. The characterized Escherichia phage myPSH1131 was found to have broad host range activity against E. coli pathogens and in vivo studies showed that multiple doses are required for effective treatment.

Purpose. The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem- and colistin-resistance in two areas in Tamil Nadu, India. Methodology. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem- and colistin-resistance. Further, resistance genes bla NDM-1, bla OXA-48-like, bla IMP, bla VIM, bla KPC, mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed. Results. A total of 89 bacterial isolates were studied which included Escherichia coli (n=43), Klebsiella pneumoniae (n=18), Pseudomonas aeruginosa (n=10), Enterobacter cloacae (n=6), Acinetobacter baumannii (n=5), Klebsiella oxytoca (n=4), Proteus mirabilis (n=2) and Salmonella paratyphi (n=1). MIC testing showed that 58/89 (65 %) and 29/89 (32 %) isolates were resistant to meropenem and colistin, respectively, whereas 27/89 (30 %) isolates were resistant to both antibiotics. Escherichia coli, K. pneumoniae, K. oxytoca, Pseudomonas aeruginosa, and Enterobacter cloacae isolates were bla NDM-1-positive (n=20). Some strains of Escherichia coli, K. pneumoniae and K. oxytoca were bla OXA-181-positive (n=4). Class 1, 2 and 3 integrons were found in 24, 20 and 3 isolates, respectively. Nine NDM-1-positive Escherichia coli strains could transfer carbapenem resistance via plasmids to susceptible Escherichia coli AB1157. Meropenem and colistin showed synergy in 10/20 (50 %) isolates by 24 h time-kill studies. Conclusion. Our results highlight the distribution of carbapenem- and colistin-resistance in Gram-negative bacteria isolated from the Tamil Nadu region in South India.