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Acinetobacter pittii is an important pathogen causing nosocomial infection worldwide. In this study, a multidrug-resistant A. pittii ABC38 was used as host bacterium to isolate the lytic phage vB_ApiP_XC38. The biological characteristics of vB_ApiP_XC38 were studied and the genome was sequenced and analyzed. vB_ApiP_XC38 belonged to Podoviridae family. The phage had double-stranded genome, which comprised 79,328 bp with 39.58% G+C content displaying very low similarity (

We report a case of a 63-year-old female patient who developed a recurrent urinary tract infection (UTI) with extensively drug-resistant Klebsiella pneumoniae (ERKp). In the initial two rounds of phage therapy, phage resistant mutants developed within days. Although ERKp strains were completely resistant to sulfamethoxazole-trimethoprim, the combination of sulfamethoxazole-trimethoprim with the phage cocktail inhibited the emergence of phage resistant mutant in vitro, and the UTI of patient was successfully cured by this combination. Thus, we propose that non-active antibiotic and bacteriophage synergism (NABS) might be an alternative strategy in personalized phage therapy.

Staphylococcus aureus (S. aureus), considered as a common foodborne pathogenic microorganism, usually causes food poisoning and various infectious diseases. Therefore, development of rapid and accurate bacterial detection method is the key to preventing food poisoning and achieving early diagnosis and treatment of various infectious diseases caused by S. aureus. Biolayer interferometry (BLI) technology is a novel technique of label-free optical analysis for real-time monitoring of biomolecular interactions. The C54A mutation induced the lytic activity loss of phage lysin LysGH15 but retained the capacity for specific recognizing and binding S. aureus. In this study, a novel method for the detection of S. aureus was established using the C54A mutant LysGH15 as the receptor in combination with BLI. Using this BLI-based method, S. aureus whole cells could be directly assayed and the limit of detection was 13 CFU/mL with a binding time of 12 min. Because the C54A mutant LysGH15 recognizes S. aureus with very high specificity, the method can exclude potential interference from other bacterial species. In addition, this method could also distinguish between viable and dead S. aureus. Moreover, S. aureus was successfully detected in ice cubes and light soy sauce by using this method. Collectively, these results indicate that the LysGH15-based BLI method can be used as an efficient and reliable diagnostic tool in the field of food safety and other related fields for the rapid, sensitive, label-free, and real-time detection of S. aureus.

Bacteriophages have been recently revisited as an alternative biocontrol tool due to the limitations of antibiotic treatment. In this study, we reported on the biological characteristics and genomic information of vB_KpnS_GH-K3 (abbreviated as GH-K3), a Klebsiella phage of the Siphoviridae family, which was previously isolated from a hospital sewage system. One-step growth curve analysis indicated that the burst size of GH-K3 was 291 PFU/cell. GH-K3 maintained a stable titer in a broad range of pH values (6-10) and temperature (up to 50 °C). Based on bioinformatics analysis, GH-K3 comprises of 49,427 bp containing a total of 77 open reading frames (ORFs), which share high degree of nucleotide similarity and close evolutionary relationships with at least 12 other Klebsiella phages. Of note, GH-K3 gp32 was identified as a unique ORF. The major segment of gp32 sequence at the C-terminus (residues 351-907) was found highly variable as determined by its mismatch with the nucleotide and protein sequences available at NCBI database. Furthermore, HHpred analysis indicated that GH-K3 gp32 contains three domains (PDB ID: 5W6S_A, 3GQ8_A and 1BHE_A) similar to depolymerase (depoKP36) of Klebsiella phage KP36 suggestive of a potential depolymerase activity during host receptor-binding in the processes of phage infection. Altogether, the current data revealed a novel putative depolymerase-like protein which is most likely to play an important role in phage-host interaction.

Staphylococcus aureus is one of the most important pathogens causing rabbit necrotizing pneumonia and brings huge economic losses to rabbit production. This study investigated the preventive effect of a phage on rabbit necrotizing pneumonia caused by S. aureus. S. aureus S6 was isolated from the lungs of rabbits suffering necrotizing pneumonia and identified. A novel phage named VB-SavM-JYL01 was isolated by using S. aureus S6 as a host and showed a broader host range than the phages GH15 and K. The genome of VB-SavM-JYL01 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. A single intranasal administration of VB-SavM-JYL01 (3 × 109 PFU) could effectively improve the survival rate at 48 h to 90% (9/10) compared with the survival rate of 10% and 80% observed with the PBS or linezolid treatment, respectively. The bacterial count in the lungs of rabbits treated with the phage VB-SavM-JYL01 was 4.18 × 104 CFU/g at 24 h, which was significantly decreased compared to that of rabbits treated with PBS (7.38 × 107 CFU/g) or linezolid (3.12 × 105 CFU/g). The phage treatment significantly alleviated lung tissue damage. The levels of total proteins, Panton-Valentine leukocidin (PVL), alpha-toxin (Hla) and cytokines in the lungs of the rabbits treated with the phage were significantly lower than those of the rabbits treated with PBS and similar to those of the rabbits treated with linezolid. These data demonstrate the potential utility of phage as an alternative for preventing rabbit necrotizing pneumonia caused by S. aureus.

Porcine contagious pleuropneumonia is a highly fatal respiratory disease that is caused by Actinobacillus pleuropneumoniae (APP) and results in tremendous economic losses for the pig breeding industry worldwide. Previous studies have demonstrated that Propionibacterium acnes (PA) could effectively prevent APP infection in mice and pigs. The humoral immune response played a primary role during this process and anti-PA antibody could mediate macrophages to kill the bacteria. However, the role of neutrophils in this process is currently unknown. In this study, mice were injected with cyclophosphamide to deplete neutrophils and then passively immunized with anti-PA serum or negative serum. Mice were subsequently challenged with APP serotype 1. The results showed that the mice exhibited less bacterial colonization, less lung damage, and a high survival rate, which were immunized with the anti-PA antibody whether neutrophils were depleted or not. Worse still, the presence of neutrophils increased the damage to the mice after challenge. These results suggest that the activity of the anti-PA antibody against APP infection was independent of neutrophils. These findings have important significance for understanding the mechanisms of humoral immunity conferred by heterologous immunization and lay a good foundation for preventing APP infection.

The bacterial pathogen Klebsiella pneumoniae causes urinary tract infections in immunocompromised patients. Generally, the overuse of antibiotics contributes to the potential development and the spread of antibiotic resistance. In fact, certain strains of K. pneumoniae are becoming increasingly resistant to antibiotics, making infection by these strains more difficult to treat. The use of bacteriophages to control pathogens may offer a non-antibiotic-based approach to treat multidrug-resistant (MDR) infections. However, a detailed understanding of phage–host interactions is crucial in order to explore the potential success of phage-therapy for treatment. In this study, we investigated the molecular epidemiology of nine carbapenemase-producing K. pneumoniae isolates from a local hospital in Shanghai, China. All strain isolates belong to sequence type 11 (ST11) and harbor the blaKPC-2 gene. The S1-PFGE (S1 nuclease pulsed field gel electrophoresis) pattern of the isolates did not show any relationship to the multilocus sequence typing (MLST) profiles. In addition, we characterized phage 117 and phage 31 and assessed the potential application of phage therapy in treating K. pneumoniae infections in vitro. The results of morphological and genomic analyses suggested that both phages are affiliated to the T7 virus genus of the Podoviridae family. We also explored phage–host interactions during growth in both planktonic cells and biofilms. The phages’ heterogeneous lytic capacities against K. pneumoniae strains were demonstrated experimentally. Subsequent culture and urine experiments with phage 117 and host Kp36 initially demonstrated a strong lytic activity of the phages. However, rapid regrowth was observed following the initial lysis which suggests that phage resistant mutants were selected in the host populations. Additionally, a phage cocktail (117 + 31) was prepared and investigated for antimicrobial activity. In Luria Broth (LB) cultures, we observed that the cocktail showed significantly higher antimicrobial activity than phage 117 alone, but this was not observed in urine samples. Together, the results demonstrate the potential therapeutic value of phages in treating K. pneumoniae urinary tract infections.

Klebsiella pneumoniae (K. pneumoniae) spp. are important nosocomial and community-acquired opportunistic pathogens, which cause various infections. We observed that K. pneumoniae strain K7 abruptly mutates to rough-type phage-resistant phenotype upon treatment with phage GH-K3. In the present study, the rough-type phage-resistant mutant named K7RR showed much lower virulence than K7. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis indicated that WcaJ and two undefined glycosyltransferases (GTs)- named GT-1, GT-2- were found to be down-regulated drastically in K7RR as compared to K7 strain. GT-1, GT-2, and wcaJ are all located in the gene cluster of capsular polysaccharide (CPS). Upon deletion, even of single component, of GT-1, GT-2, and wcaJ resulted clearly in significant decline of CPS synthesis with concomitant development of GH-K3 resistance and decline of virulence of K. pneumoniae, indicating that all these three GTs are more likely involved in maintenance of phage sensitivity and bacterial virulence. Additionally, K7RR and GT-deficient strains were found sensitive to endocytosis of macrophages. Mitogen-activated protein kinase (MAPK) signaling pathway of macrophages was significantly activated by K7RR and GT-deficient strains comparing with that of K7. Interestingly, in the presence of macromolecular CPS residues (>250 KD), K7(ΔGT-1) and K7(ΔwcaJ) could still be bounded by GH-K3, though with a modest adsorption efficiency, and showed minor virulence, suggesting that the CPS residues accumulated upon deletion of GT-1 or wcaJ did retain phage binding sites as well maintain mild virulence. In brief, our study defines, for the first time, the potential roles of GT-1, GT-2, and WcaJ in K. pneumoniae in bacterial virulence and generation of rough-type mutation under the pressure of bacteriophage.

Aerococcus viridans is an opportunistic pathogen that is clinically associated with various human and animal diseases. In this study, the first identified A. viridans phage, vB_AviM_AVP (abbreviated as AVP), was isolated and studied. AVP belongs to the family Myoviridae. AVP harbors a double-stranded DNA genome with a length of 133,806 bp and a G + C content of 34.51%. The genome sequence of AVP showed low similarity (&lt, 1% identity) to those of other phages, bacteria, or other organisms in the database. Among 165 predicted open reading frames (ORFs), there were only 69 gene products exhibiting similarity (&le, 65% identity) to proteins of known functions in the database. In addition, the other 36 gene products did not match any viral or prokaryotic sequences in any publicly available database. On the basis of the putative functions of the ORFs, the genome of AVP was divided into three modules: nucleotide metabolism and replication, structural components, and lysis. A phylogenetic analysis of the terminase large subunits and capsid proteins indicated that AVP represents a novel branch of phages. The observed characteristics of AVP indicate that it represents a new class of phages.

Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we observed the ability of the phage lysin LysGH15 to eliminate staphylococcal planktonic cells and biofilms formed by Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis All these strains were sensitive to LysGH15, showing reductions in bacterial counts of approximately 4 log units within 30 min after treatment with 20 μg/ml of LysGH15, and the MICs ranged from 8 μg/ml to 32 μg/ml. LysGH15 efficiently prevented biofilm formation by the four staphylococcal species at a dose of 50 μg/ml. At a higher dose (100 μg/ml), LysGH15 also showed notable disrupting activity against 24-h and 72-h biofilms formed by S. aureus and coagulase-negative species. In the in vivo experiments, a single intraperitoneal injection of LysGH15 (20 μg/mouse) administered 1 h after the injection of S. epidermidis at double the minimum lethal dose was sufficient to protect the mice. The S. epidermidis cell counts were 4 log units lower in the blood and 3 log units lower in the organs of mice 24 h after treatment with LysGH15 than in the untreated control mice. LysGH15 reduced cytokine levels in the blood and improved pathological changes in the organs. The broad antistaphylococcal activity exerted by LysGH15 on planktonic cells and biofilms makes LysGH15 a valuable treatment option for biofilm-related or non-biofilm-related staphylococcal infections.IMPORTANCE Most staphylococcal species are major causes of health care- and community-associated infections. In particular, Staphylococcus aureus is a common and dangerous pathogen, and Staphylococcus epidermidis is a ubiquitous skin commensal and opportunistic pathogen. Treatment of infections caused by staphylococci has become more difficult because of the emergence of multidrug-resistant strains as well as biofilm formation. In this study, we found that all tested S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis strains were sensitive to the phage lysin LysGH15 (MICs ranging from 8 to 32 μg/ml). More importantly, LysGH15 not only prevented biofilm formation by these staphylococci but also disrupted 24-h and 72-h biofilms. Furthermore, the in vivo efficacy of LysGH15 was demonstrated in a mouse model of S. epidermidis bacteremia. Thus, LysGH15 exhibits therapeutic potential for treating biofilm-related or non-biofilm-related infections caused by diverse staphylococci.

Bacteriophage can be used as an alternative or complementary therapy to antibiotics for treating multidrug-resistant bacterial infections. However, the rapid emergence of resistant host variants during phage treatment has limited its therapeutic applications. In this study, a potential phage-resistant mechanism of Klebsiella pneumoniae was revealed. After phage GH-K3 treatment, a smooth-type colony, named K7R(B), was obtained from the K. pneumoniae K7 culture. Treatment with IO(4)(−) and/or proteinase K indicated that polysaccharides of K7 played an important role in phage recruitment, and protein receptors on K7 were essential for effective infection by GH-K3. Differences in protein expression between K7 and K7R(B) were quantitatively analyzed by liquid chromatography-tandem mass spectrometry. Among differentially expressed proteins, OmpC, OmpN, KPN_02430, and OmpF were downregulated significantly in K7R(B). trans-Complementation of OmpC in K7R(B) conferred rapid adsorption and sensitivity to GH-K3. In contrast, a single-base deletion mutation of ompC in K7, which resulted in OmpC silencing, led to lower adsorption efficiency and resistance to GH-K3. These assays proved that OmpC is the key receptor-binding protein for GH-K3. In addition, the native K. pneumoniae strains KPP14, KPP27, and KPP36 showed low or no sensitivity to GH-K3. However, these strains became more sensitive to GH-K3 after their native receptors were replaced by OmpC of K7, suggesting that OmpC(K7) was the most suitable receptor for GH-K3. This study revealed that K7R(B) became resistant to GH-K3 due to gene mutation of ompC and that OmpC of K7 is essential for effective infection by GH-K3. IMPORTANCE With increased incidence of multidrug-resistant (MDR) bacterial strains, phages have regained attention as promising potential antibacterial agents. However, the rapid emergence of resistant variants during phage treatment has limited the therapeutic applications of phage. According to our trans-complementation, ompC mutation, and phage adsorption efficiency assays, we identified OmpC as the key receptor-binding protein (RBP) for phage GH-K3, which is essential for effective infection. This study revealed that the phage secondary receptor of K. pneumoniae, OmpC, is the essential RBP not only for phage infecting Gram-negative bacteria, such as Escherichia coli and Salmonella, but also for K. pneumoniae.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

Phage-derived lysins can hydrolyse bacterial cell walls and show great potential for combating Gram-positive pathogens. In this study, the potential of LysEF-P10, a new lysin derived from a isolated Enterococcus faecalis phage EF-P10, as an alternative treatment for multidrug-resistant E. faecalis infections, was studied. LysEF-P10 shares only 61% amino acid identity with its closest homologues. Four proteins were expressed: LysEF-P10, the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain (LysEF-P10C), the putative binding domain (LysEF-P10B), and a fusion recombination protein (LysEF-P10B-green fluorescent protein). Only LysEF-P10 showed highly efficient, broad-spectrum bactericidal activity against E. faecalis. Several key functional residues, including the Cys-His-Asn triplet and the calcium-binding site, were confirmed using 3D structure prediction, BLAST and mutation analys. We also found that calcium can switch LysEF-P10 between its active and inactive states and that LysEF-P10B is responsible for binding E. faecalis cells. A single administration of LysEF-P10 (5 μg) was sufficient to protect mice against lethal vancomycin-resistant Enterococcus faecalis (VREF) infection, and LysEF-P10-specific antibody did not affect its bactericidal activity or treatment effect. Moreover, LysEF-P10 reduced the number of Enterococcus colonies and alleviated the gut microbiota imbalance caused by VREF. These results indicate that LysEF-P10 might be an alternative treatment for multidrug-resistant E. faecalis infections.

Enterococcus faecalis is becoming an increasingly important opportunistic pathogen worldwide, especially because it can cause life-threatening nosocomial infections. Treating E. faecalis infections has become increasingly difficult because of the prevalence of multidrug-resistant E. faecalis strains. Because bacteriophages show specificity for their bacterial hosts, there has been a growth in interest in using phage therapies to combat the rising incidence of multidrug-resistant bacterial infections. In this study, we isolated a new lytic phage, EF-P29, which showed high efficiency and a broad host range against E. faecalis strains, including vancomycin-resistant strains. The EF-P29 genome contains 58,984 bp (39.97% G+C), including 101 open reading frames, and lacks known putative virulence factors, integration-related proteins or antibiotic resistance determinants. In murine experiments, the administration of a single intraperitoneal injection of EF-P29 (4 × 105 PFU) at 1 h after challenge was sufficient to protect all mice against bacteremia caused by infection with a vancomycin-resistant E. faecalis strain (2 × 109 CFU/mouse). E. faecalis colony counts were more quickly eliminated in the blood of EF-P29-protected mice than in unprotected mice. We also found that exogenous E. faecalis challenge resulted in enrichment of members of the genus Enterococcus (family Enterococcaceae) in the guts of the mice, suggesting that it can enter the gut and colonize there. The phage EF-P29 reduced the number of colonies of genus Enterococcus and alleviated the gut microbiota imbalance that was caused by E. faecalis challenge. These data indicate that the phage EF-P29 shows great potential as a therapeutic treatment for systemic VREF infection. Thus, phage therapies that are aimed at treating opportunistic pathogens are also feasible. The dose of phage should be controlled and used at the appropriate level to avoid causing imbalance in the gut microbiota.

The lysin LysGH15, derived from the staphylococcal phage GH15, exhibits a wide lytic spectrum and highly efficient lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). Here, we found that LysGH15 did not induce resistance in MRSA or methicillin-sensitive S. aureus (MSSA) strains after repeated treatment. Although LysGH15 triggered the generation of LysGH15-specific antibodies in mice, these antibodies did not block lytic activity in vitro (nor the binding capacity of LysGH15). More importantly, when the antibody titre was highest in mice immunized with LysGH15, a single intravenous injection of LysGH15 was sufficient to protect mice against lethal infection with MRSA. These results indicated that LysGH15-specific antibodies did not affect the killing efficiency of LysGH15 against MRSA in vitro or in vivo. LysGH15 also reduced pro-inflammatory cytokines in mice with lethal infections. Furthermore, a high-dose LysGH15 injection did not cause significant adverse effects or pathological changes in the main organs of treated animals. These results provide further evidence for the administration of LysGH15 as an alternative strategy for the treatment of infections caused by MRSA.

Inulin is a natural fructose polymer that can be used as a fat substitute in foods such as dairy products and bakery products and is often added to food products due to its effect on the regulation of intestinal flora (also known as the prebiotic effect). However, there are few studies exploring whether there are functional differences between fructose species with different degrees of polymerization (DP). Therefore, we focused on the separation of fructose species with different DP and their different effects on the balance of intestinal flora. First, the fractional precipitation method was used to separate short-chain inulin (DP:2-9), medium-chain inulin (DP:10-23), and long-chain inulin (DP:23 and above). Then, male C57BL/6 mice were randomly distributed into four groups and fed with a high-fat diet(HFD), a high-fat diet with added short-chain inulin(SCI), a high-fat diet with added medium-chain inulin(MCI), or a high-fat diet with added long-chain inulin(LCI) for two weeks. Finally, RT-PCR was used to detect the relative abundance of specific bacteria after this feeding course. The results showed that the abundance of common probiotics increased, and some harmful bacteria reduced after SCI, MCI, and HCI treatment. As the short-chain inulin has the strongest effect on improving the balance of intestinal flora, it may be a promising treatment option for patients with obesity, fatty liver, diabetes or other gastrointestinal issues.

Pneumonia is one of the most prevalent Staphylococcus aureus -mediated diseases, and the treatment of this infection is becoming challenging due to the emergence of multidrug-resistant S. aureus , especially methicillin-resistant S. aureus (MRSA) strains. It has been reported that LysGH15, the lysin derived from phage GH15, displays high efficiency and a broad lytic spectrum against MRSA and that apigenin can markedly diminish the alpha-hemolysin of S. aureus . In this study, the combination therapy of LysGH15 and apigenin was evaluated in vitro and in a mouse S. aureus pneumonia model. No mutual adverse influence was detected between LysGH15 and apigenin in vitro . In animal experiments, the combination therapy showed a more effective treatment effect than LysGH15 or apigenin monotherapy ( P < 0.05). The bacterial load in the lungs of mice administered the combination therapy was 1.5 log units within 24 h after challenge, whereas the loads in unprotected mice or mice treated with apigenin or LysGH15 alone were 10.2, 4.7, and 2.6 log units, respectively. The combination therapy group showed the best health status, the lowest ratio of wet tissue to dry tissue of the lungs, the smallest amount of total protein and cells in the lung, the fewest pathological manifestations, and the lowest cytokine level compared with the other groups ( P < 0.05). With regard to its better protective efficacy, the combination therapy of LysGH15 and apigenin exhibits therapeutic potential for treating pneumonia caused by MRSA. This paper reports the combination therapy of lysin and natural products derived from traditional Chinese medicine.