Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection.

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

vB_EcoM-P896 coliphage isolated from duck sewage can lyse both intestinal pathogenic Escherichia coli and extraintestinal pathogenic E. coli.

Pathogenic Escherichia coli strains cause diseases in both humans and animals. The limiting factors to prevent as well as control infections from pathogenic E. coli strains are their pathotypes, serotypes, and drug resistance. Herein, a bacteriophage (vB_EcoM-P896) has been isolated from duck sewage. Furthermore, aside from targeting intestinal pathogenic E. coli strains like enteropathogenic E. coli, Shiga toxin-producing E. coli, entero-invasive E. coli, and enteroaggregative E. coli, vB_EcoM-P896 can cause lysis in extraintestinal pathogenic E. coli strains such as avian pathogenic E. coli. Stability analysis revealed that vB_EcoM-P896 was stable under the following conditions: temperature, 4℃-50℃; pH, 3-11. The sequencing of the vB_EcoM-P896 genome was conducted utilizing an HiSeq system (Illumina, San Diego, CA) and subjected to de novo assembling with the aid of Spades 3.11.1. The characteristics of the DNA genome were as follows: size, 170,656 bp; GC content, 40.4%; the number of putative coding regions, 294. Transmission electron microscopy analysis of morphology and genome analysis revealed that the phage vB_EcoM-P896 belonged to the order Caudovirales and the family Myoviridae. The pan-genome analysis of vB_EcoM-P896 was divided into two levels. The first level involved the analysis of 91 strains of muscle tail phages, which were mainly divided into 5 groups. The second level involved the analysis of 24 strains of myophage with high homology. Of the 1480 gene clusters, 23 were shared core genes. Neighbor-joining phylogenetic trees were constructed using the Poisson model with MEGA6.0 based on the conserved sequences of phage proteins, the amino acid sequence of the terminase large subunit, and tail fibrin. Further analysis revealed that vB_EcoM-P896 was a typical T4-like potent phage with potential clinical applications.

Antimicrobial resistance survey and whole-genome analysis of nosocomial P. Aeruginosa isolated from eastern Province of China in 2016-2021.

BACKGROUND: Pseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death. METHODS: From 2016 to 2021, 103 strains of P. aeruginosa were isolated from hospitals and 20 antibiotics were used for antimicrobial susceptibility determination. Using next-generation genome sequencing technology, these strains were sequenced and analyzed in terms of serotypes, ST types, and resistance genes for epidemiological investigation. RESULTS: The age distribution of patients ranged from 10 days to 94 years with a median age of 69 years old. The strains were mainly isolated from sputum (72 strains, 69.9%) and blood (14 strains, 13.6%). The size of these genomes ranged from 6.2 Mb to 7.4 Mb, with a mean value of 6.5 Mb. In addition to eight antibiotics that show inherent resistance to P. aeruginosa, the sensitivity rates for colistin, amikacin, gentamicin, ceftazidime, piperacillin, piperacillin-tazobactam, ciprofloxacin, meropenem, aztreonam, imipenem, cefepime and levofloxacin were 100%, 95.15%, 86.41%, 72.82%, 71.84%, 69.90%, 55.34%, 52.43%, 50.49%, 50.49%, 49.51% and 47.57% respectively, and the carriage rate of MDR strains was 30.69% (31/101). Whole-genome analysis showed that a total of 50 ST types were identified, with ST244 (5/103) and ST1076 (4/103) having a more pronounced distribution advantage. Serotype predictions showed that O6 accounted for 29.13% (30/103), O11 for 23.30% (24/103), O2 for 18.45% (19/103), and O1 for 11.65% (12/103) of the highest proportions. Notably, we found a significantly higher proportion of ExoU in P. aeruginosa strains of serotype O11 than in other cytotoxic exoenzyme positive strains. In addition to this, a total of 47 crpP genes that mediate resistance to fluoroquinolones antibiotics were found distributed on 43 P. aeruginosa strains, and 10 new variants of CrpP were identified, named 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.40, 1.41 and 7.1. CONCLUSIONS: We investigated the antibiotic susceptibility of clinical isolates of P. aeruginosa and genomically enriched the diversity of P. aeruginosa for its prophylactic and therapeutic value.

Sifting through the core-genome to identify putative cross-protective antigens against Riemerella anatipestifer.

Infectious serositis of ducks, caused by Riemerella anatipestifer, is one of the main infectious diseases that harm commercial ducks. Whole-strain-based vaccines with no or few cross-protection were observed between different serotypes of R. anatipestifer, and so far, control of infection is hampered by a lack of effective vaccines, especially subunit vaccines with cross-protection. Since the concept of reverse vaccinology was introduced, it has been widely used to screen for protective antigens in important pathogens. In this study, pan-genome binding reverse vaccinology, an emerging approach to vaccine candidate screening, was used to screen for cross-protective antigens against R. anatipestifer. Thirty proteins were identified from the core-genome as potential cross-protective antigens. Three of these proteins were recombinantly expressed, and their immunoreactivity with five antisera (anti-serotypes 1, 2, 6, 10, and 11) was demonstrated by Western blotting. Our study established a method for high-throughput screening of cross-protective antigens against R. anatipestifer in silico, which will lay the foundation for the development of a cross-protective subunit vaccine controlling R. anatipestifer infection. KEY POINTS: • Pan-genome binding reverse vaccine approach was first established in R. anatipestifer to screen for subunit vaccine candidates. • Thirty potential cross-protective antigens against R. anatipestifer were identified by this method. • The reliability of the method was verified preliminarily by the results of Western blotting of three of these potential antigens.

Machine learning combined with MALDI-TOF MS has the potential ability to identify serotypes of the avian pathogen Riemerella anatipestifer.

AIM: Combining MALDI-TOF MS and machine learning to establish a new rapid method to identify two important serotypes of Rimerella anatipestifer. METHODS AND RESULTS: MALDI-TOF MS was performed on 115 R. anatipestifer strains (serotype 1, serotype 2, and other serotypes) to explore its ability to identify serotypes of R. anatipestifer. Raw spectral data were generated in diagnostic mode; these data were preprocessed, clustered, and analysed using principal component analysis. The results indicated that MALDI-TOF MS completely differentiated serotype 1 from serotype 2 of R. anatipestifer; the potential serotype-associated m/z loci are listed. Furthermore, Random Forest and Support Vector Machine were used for modelling to identify the two important serotypes, and the results of cross-validation indicated that they had ∼80% confidence to make the right classification. CONCLUSION: We proved that MALDI-TOF MS can differentiate serotype 1 from serotype 2 of R. anatipestifer. Additionally, the identification models established in this study have high confidence to screen out these two important serotypes from other serotypes.

Identification of Subunits for Novel Universal Vaccines against Three Predominant Serogroups and the Emerging O145 among Avian Pathogenic Escherichia coli by Pan-RV Pipeline.

Avian pathogenic Escherichia coli, a causative agent of avian colibacillosis, has been causing serious economic losses in the poultry industry. The increase in multidrug-resistant isolates and the complexity of the serotypes of this pathogen, especially the recently reported emergence of a newly predominant serogroup of O145, make the control of this disease difficult. To address this challenge, a high-throughput screening approach, called Pan-RV (Reverse vaccinology based on pangenome analysis), is proposed to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened from the core genome of 127 Avian pathogenic Escherichia coli (APEC) genomes, and six were verified by Western blots using antisera. Overall, our research will provide a foundation for the development of an APEC subunit vaccine against avian colibacillosis. Given the exponential growth of whole-genome sequencing (WGS) data, our Pan-RV pipeline will make screening of bacterial vaccine candidates inexpensive, rapid, and efficient. IMPORTANCE With the emergence of drug resistance and the newly predominant serogroup O145, the control of Avian pathogenic Escherichia coli is facing a serious challenge; an efficient immunological method is urgently needed. Here, for the first time, we propose a high-throughput screening approach to search for universal protective antigens against the three traditional serogroups and the newly emerged O145. Importantly, using this approach, a total of 61 proteins regarded as probable antigens against the four important serogroups were screened, and three were shown to be immunoreactive with all antisera (covering the four serogroups), thereby providing a foundation for the development of APEC subunit vaccines against avian colibacillosis. Further, our Pan-RV pipeline will provide immunological control strategies for pathogens with complex and variable genetic backgrounds such as Escherichia coli and will make screening of bacterial vaccine candidates more inexpensive, rapid, and efficient.

Isolation, characterization, and genome analysis of bacteriophage P929 that could specifically lyase the KL19 capsular type of Klebsiella pneumoniae.

In recent years, Klebsiella pneumoniae has caused an increase in the number of serious infections associated with pneumonia, septicemia, urinary tract infections, and pyogenic liver abscess. In this study, a phage P929, isolated from hospital sewage in Jiangsu, could specifically infect K. pneumoniae KL19 capsular type by forming plaques with a translucent halo that expanded over time. Phage P929 with a multiplicity of infection (MOI) of 0.1 produced the highest phage titer. According to a one-step growth curve experiment, the latent time period of phage P929 was 25 min, and the burst size was about 156 phage particles/cell. The sensitivity tests confirmed that P929 was stable at temperatures ranging from 4 to 50 °C and pH 3 to 11. Based on morphological observation and phylogenetic analysis, phage P929 could be assigned to a new species in the genus Drulisvirus of the subfamily Slopekvirinae in the family Autographiviridae. According to genome analysis, phage P929 was 44,764 bp in size with 53.66% G + C content, encoding 57 proteins or coding sequences (117-3699 bp in length). Phage P929 showed potential antibacterial activity on planktonic cells and biofilm. After 120 min, the OD600 values of five phage-treated groups were basically reached zero compared to the untreated group, and the antibacterial activity of P929 was still detectable within 390 min. In anti-biofilm tests, phage P929 at an MOI of 1 significantly reduced the biofilm formation of K. pneumoniae in 48 h. These results suggest that phage P929 may be used to treat carbapenem-resistant and biofilm-forming K. pneumonia in clinical settings.

O145 may be emerging as a predominant serogroup of Avian pathogenic Escherichia coli (APEC) in China.

Among the numerous serotypes of Avian pathogenic Escherichia coli (APEC), O1, O2 and O78 have long been considered the predominant serogroups. O145, a pivotal serogroup in non-O157 Shiga toxin-producing Escherichia coli, has never been considered an important serogroup among APEC. The prevalence of APEC O145 was determined from the results of molecular serogrouping based on 42 sequenced isolates from Jiangsu and Guangxi Provinces in China. After realizing the potential importance of O145, 224 APEC isolates isolated from Jiangsu, Guangxi, Anhui, Shandong, Henan, Yunnan and Fujian provinces were screened using PCR amplification. The results showed that the proportion of O145 detected was 37.9 % (85/224), which was higher than those of the three traditional APEC serogroups. The virulence evaluation experiment showed that this serogroup may have stronger pathogenicity. Here, we report for the first time that O145 may be emerging as a predominant serogroup of APEC in China. The possible reasons for its prevalence and oversight were analyzed through genomic analysis. Furthermore, pangenome analysis with STEC O145 was performed to assess the potential threat to humans. The discovery of the ubiquity of O145 may not be coincidental, which may also account for the failure of vaccines that target the three major serogroups. Therefore, this newly predominant serogroup should be paid more attention and the focus should not be limited to the so-called three major APEC serogroups.

Combining pangenome analysis to identify potential cross-protective antigens against avian pathogenic Escherichia coli.

RESEARCH HIGHLIGHTSPan-RV analysis was used for the first time in the discovery of APEC-protective proteins.A total of 53 potential protective proteins were screened out.Four proteins were verified as potential vaccine candidates using western blotting.