Construction of programmed time-released multifunctional hydrogel with antibacterial and anti-inflammatory properties for impaired wound healing.

The successful reprogramming of impaired wound healing presents ongoing challenges due to the impaired tissue microenvironment caused by severe bacterial infection, excessive oxidative stress, as well as the inappropriate dosage timing during different stages of the healing process. Herein, a dual-layer hydrogel with sodium alginate (SA)-loaded zinc oxide (ZnO) nanoparticles and poly(N-isopropylacrylamide) (PNIPAM)-loaded Cu5.4O ultrasmall nanozymes (named programmed time-released multifunctional hydrogel, PTMH) was designed to dynamically regulate the wound inflammatory microenvironment based on different phases of wound repairing. PTMH combated bacteria at the early phase of infection by generating reactive oxygen species through ZnO under visible-light irradiation with gradual degradation of the lower layer. Subsequently, when the upper layer was in direct contact with the wound tissue, Cu5.4O ultrasmall nanozymes were released to scavenge excessive reactive oxygen species. This neutralized a range of inflammatory factors and facilitated the transition from the inflammatory phase to the proliferative phase. Furthermore, the utilization of Cu5.4O ultrasmall nanozymes enhanced angiogenesis, thereby facilitating the delivery of oxygen and nutrients to the impaired tissue. Our experimental findings indicate that PTMHs promote the healing process of diabetic wounds with bacterial infection in mice, exhibiting notable antibacterial and anti-inflammatory properties over a specific period of time.

Hyaluronidase-Responsive Bactericidal Cryogel for Promoting Healing of Infected Wounds: Inflammatory Attenuation, ROS Scavenging, and Immune Regulation.

Wounds infected with multidrug-resistant (MDR) bacteria are increasingly threatening public health and challenging clinical treatments because of intensive bacterial colonization, excessive inflammatory responses, and superabundant oxidative stress. To overcome this malignant burden and promote wound healing, a multifunctional cryogel (HA/TA2/KR2) composed of hyaluronic acid (HA), tannic acid (TA), and KR-12 peptides is designed. The cryogel exhibited excellent shape-memory properties, strong absorption performance, and hemostatic capacity. In vitro experiments demonstrated that KR-12 in the cryogel can be responsively released by stimulation with hyaluronidase produced by bacteria, reaching robust antibacterial activity against Escherichia coli (E. coli), MDR Pseudomonas aeruginosa (MDR-PA), and methicillin-resistant Staphylococcus aureus (MRSA) by disrupting bacterial cell membranes. Furthermore, the synergetic effect of KR-12 and TA can efficiently scavenge ROS and decrease expression of pro-inflammatory cytokines (tumor necrosis factor (TNF)-α & interleukin (IL)-6), as well as modulate the macrophage phenotype toward the M2 type. In vivo animal tests indicated that the cryogel can effectively destroy bacteria in the wound and promote healing process via accelerating angiogenesis and re-epithelialization. Proteomic analysis revealed the underlying mechanism by which the cryogel mainly reshaped the infected wound microenvironment by inhibiting the Nuclear factor kappa B (NF-κB) signaling pathway and activating the Janus kinase-Signal transducer and activator of transcription (JAK-STAT6) signaling pathway. Therefore, the HA/TA2/KR2 cryogel is a promising dressing candidate for MDR bacteria-infected wound healing.

The m6A reader YTHDF2 alleviates the inflammatory response by inhibiting IL-6R/JAK2/STAT1 pathway-mediated high-mobility group box-1 release.

Background: Sepsis is a common severe complication in major burn victims and is characterized by a dysregulated systemic response to inflammation. YTH domain family 2 (YTHDF2), a well-studied N6-methyladenosine (m6A) reader that specifically recognizes and binds to m6A-modified transcripts to mediate their degradation, is connected to pathogenic and physiological processes in eukaryotes, but its effect on sepsis is still unknown. We aimed to discover the effects and mechanisms of YTHDF2 in sepsis. Methods: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot analyses were used to measure the expression of YTHDF2, the interleukin 6 receptor (IL-6R), high-mobility group box-1 (HMGB1), Janus kinase 2 (JAK2) and signal transducer and activator of transcription 1 (STAT1) under different in vitro conditions. Enzyme-linked immunosorbent assays were utilized to evaluate the expression of HMGB1, IL-6, IL-1ß and tumor necrosis factor-α. To confirm that YTHDF2 specifically targets IL-6R mRNA, RNA immunoprecipitation and dual-luciferase reporter assays were performed. Finally, we utilized a mouse model of lipopolysaccharide (LPS)-induced sepsis to verify the effects of YTHDF2 in vivo. Results: According to our findings, YTHDF2 was expressed at a low level in peripheral blood mononuclear cells from septic mice and patients as well as in LPS-induced RAW264.7 cells. Overexpression of YTHDF2 alleviated the inflammatory response by inhibiting HMGB1 release and JAK2/STAT1 signalling in LPS-stimulated cells. Mechanistically, YTHDF2 suppressed JAK2/STAT1 signalling by directly recognizing the m6A-modified site in IL-6R and decreasing the stability of IL-6R mRNA, thereby inhibiting HMGB1 release. In vivo experiments showed that YTHDF2 played a protective role in septic mice by suppressing the IL-6R/JAK2/STAT1/HMGB1 axis. Conclusions: In summary, these findings demonstrate that YTHDF2 plays an essential role as an inhibitor of inflammation to reduce the release of HMGB1 by inhibiting the IL-6R/JAK2/STAT1 pathway, indicating that YTHDF2 is a novel target for therapeutic interventions in sepsis.

SVep1, a temperate phage of human oral commensal Streptococcus vestibularis.

Introduction: Bacteriophages play a vital role in the human oral microbiome, yet their precise impact on bacterial physiology and microbial communities remains relatively understudied due to the limited isolation and characterization of oral phages. To address this gap, the current study aimed to isolate and characterize novel oral phages. Methods: To achieve this, oral bacteria were isolated using a culture-omics method from 30 samples collected from healthy individuals. These bacteria were then cultured in three different types of media under both aerobic and anaerobic conditions. The samples were subsequently subjected to full-length 16S rRNA gene sequencing for analysis. Subsequently, we performed the isolation of lytic and lysogenic phages targeting all these bacteria. Results: In the initial step, a total of 75 bacterial strains were successfully isolated, representing 30 species and 9 genera. Among these strains, Streptococcus was found to have the highest number of species. Using a full-length 16S rRNA gene similarity threshold of 98.65%, 14 potential novel bacterial species were identified. In the subsequent phase, a temperate phage, which specifically targets the human oral commensal bacterium S. vestibularis strain SVE8, was isolated. The genome of S. vestibularis SVE8 consists of a 1.96-megabase chromosome, along with a 43,492-base pair prophage designated as SVep1. Annotation of SVep1 revealed the presence of 62 open reading frames (ORFs), with the majority of them associated with phage functions. However, it is worth noting that no plaque formation was observed in S. vestibularis SVE8 following lytic induction using mitomycin C. Phage particles were successfully isolated from the supernatant of mitomycin C-treated cultures of S. vestibularis SVE8, and examination using transmission electron microscopy confirmed that SVep1 is a siphovirus. Notably, phylogenetic analysis suggested a common ancestral origin between phage SVep1 and the cos-type phages found in S. thermophilus. Discussion: The presence of SVep1 may confer immunity to S. vestibularis against infection by related phages and holds potential for being engineered as a genetic tool to regulate oral microbiome homeostasis and oral diseases.

BL02, a phage against carbapenem- and polymyxin-B resistant Klebsiella pneumoniae, isolated from sewage: A preclinical study.

The emergence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) represents a threat to public health. Polymyxin-B is generally considered a last-resort antibiotic. In this study, we isolated a carbapenem- and polymyxin-B resistant K. pneumoniae phage BL02 for the first time in Southwestern China and evaluated its biological characteristics and whole-genome sequence. Polymyxin-B resistant K. pneumoniae, (CK02), was isolated from the blood of a male with severe septic shock, and phage BL02 was screened and purified from the hospital sewage. BL02 could lyse 40 out of 46 CRKP isolates (86.96%) and has high activity in the pH range of 6-10 and the temperature range of 4-55 °C. The latency period of BL02 was about 10 min and the lysis period was about 50 min. The genome results showed that BL02 was a linear dsDNA with a total length of 175,595 bp and a GC content of 41.83%. A total of 275 ORFs were predicted and no tRNA, rRNA, drug resistance genes, or virulence genes were found in the genome. Phylogenetic analysis showed that BL02 belongs to the family Straboviridae. Treatment of infected mice with two antibiotics (tigecycline or ceftazidime/avibactam) resulted in 7-day survival rates of 28.57% and 42.86%, respectively. In contrast, the survival rate of mice in the single-dose BL02-treated group was 71.43%. In summary, this preclinical study isolated a phage capable of lysing polymyxin-B resistant K. pneumoniae and validated its safety and efficacy in an in vivo model, which provides a reference for further research on controlling MDR pathogens.

Emerging Prevalence and Clinical Features of Elizabethkingia meningoseptica Infection in Southwest China: A 9-Year Retrospective Study and Systematic Review.

Background: Elizabethkingia meningoseptica infections have gradually emerged as life-threatening nosocomial infections worldwide, accompanied by increasing incidence, multidrug resistance and poor outcomes. However, the epidemiology and clinical features of E. meningoseptica infection are still limited in mainland China. Methods: Patients with E. meningoseptica infections from 2011 to 2019 in southwestern China were retrospectively analyzed. The clinical features, infection patterns and outcomes were extracted from medical records and analyzed. A comprehensive systematic review was performed in accordance with PRISMA guidelines from conception to August 23, 2021. Results: Ninety-two patients were ultimately included, with the prevalence rapidly rising from 0 in 2011 to 0.19 per 1000 inpatients in 2019. A total of 93.48% of E. meningoseptica isolates were multidrug resistant, including 100% resistance to carbapenem. Furthermore, 75% of E. meningoseptica infections were concomitant with other pathogens. The mortality of our cohort was 36.96%, with risk factors for mechanical ventilation (OR=9.51, P=0.004), male sex (OR=0.27, P=0.031) and more concomitant pathogens. After propensity score matching, central venous catheters, exposure to carbapenem and antifungal drugs, and underlying tumors were associated with E. meningoseptica infection. Sixteen articles were also summarized, with reported mortality rates ranging from 11.0% to 66.6%. Blood and respiratory tract were the common sources. Piperacillin/tazobactam, trimethoprim/sulfamethoxazole, fluoroquinolone and minocycline were the most sensitive antibiotics. Inappropriate antibiotic treatment was the most commonly reported risk factor for mortality. Conclusion: Nosocomial infection with E. meningoseptica has become an emerging problem with high mortality in southwestern China. Inappropriate antibiotic treatment and central venous catheters are risk factors for infection and death and should receive adequate attention.

LncRNA GAS5 suppresses inflammatory responses by inhibiting HMGB1 release via miR-155-5p/SIRT1 axis in sepsis.

Sepsis comprises a lethal immunologic response due to infection. Increasingly, evidence has demonstrated the important role of long non-coding RNA growth arrest-specific transcript 5 (GAS5) in the regulation of sepsis. Nevertheless, the mechanisms by which GAS5 participates in the progression of sepsis remain unclear. Our study demonstrated the role and underlying mechanism of GAS5 in regulating lipopolysaccharide (LPS)-induced inflammation. In this study, GAS5 expression was found to be markedly decreased in serum samples of sepsis patients and a sepsis mouse model, and was negatively related with HMGB1 expression. GAS5 overexpression inhibited cell inflammatory responses by decreasing HMGB1 release. Furthermore, GAS5 inhibited LPS-mediated hyperacetylation and the release of HMGB1 by increasing the expression of sirtuin1 (SIRT1). Additionally, upregulated GAS5 attenuated inflammatory responses in vitro and vivo, and the knockdown of a miR-155-5p mimic and SIRT1 rescued the effects of GAS5 upregulation. Mechanistically, GAS5 sponged miR-155-5p to upregulate SIRT1, thereby inhibiting HMGB1 acetylation and release. In conclusion, our findings indicate that GAS5 suppresses inflammatory responses by modulating the miR-155-5p/SIRT1/HMGB1 axis in sepsis, providing a novel therapeutic target for inflammation in sepsis.

Essential Fitness Repertoire of Staphylococcus aureus during Co-infection with Acinetobacter baumannii In Vivo.

Staphylococcus aureus represents a major human pathogen that is frequently involved in polymicrobial infections. However, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. In this study, we firstly performed a retrospective surveillance of 760 clinical samples and revealed a notable predominance of co-infection with S. aureus and Acinetobacter baumannii. The high-density S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) further identified a core set of genes enriched in metabolism of inorganic ions, amino acids, and carbohydrates, which are essential for infection and tissue colonization of S. aureus in the murine systemic infection model. Notably, we revealed a differential requirement of fitness factors for S. aureus in tissue-specific (liver and kidney) and infection-type-specific manner (mono- and co-infection). Co-infection with A. baumannii dramatically altered the fitness requirements of S. aureus in vivo; 49% of the mono-infection fitness genes in S. aureus strain Newman were converted to non-essential, and the functionality of ATP-binding cassette (ABC) transporters was significantly elicited during co-infection. Furthermore, the number of genes essential during co-infection (503) outnumbers the genes essential during mono-infection (362). In addition, the roles of 3 infection-type-specific genes in S. aureus during mono-infection or co-infection with A. baumannii were validated with competitive experiments in vivo. Our data indicated a high incidence and clinical relevance of S. aureus and A. baumannii co-infection, and provided novel insights into establishing antimicrobial regimens to control co-infections. IMPORTANCE Polymicrobial infections are widespread in clinical settings, which potentially correlate with increased infection severity and poor clinical outcomes. Staphylococcus aureus is a formidable human pathogen that causes a variety of diseases in polymicrobial nature. Co-infection and interaction of S. aureus have been described with limited pathogens, mainly including Pseudomonas aeruginosa, Candida albicans, and influenza A virus. Thus far, the prevalence and role of co-infectious microbes on the pathogenesis and fitness essentiality of S. aureus in vivo remain largely unknown. Understanding the polymicrobial composition and interaction, from a community and genome-wide perspective, is thus crucial to shed light on S. aureus pathogenesis strategy. Here, our findings demonstrated, for the first time, that a high incidence rate and clinical relevance of co-infection was caused by S. aureus and Acinetobacter baumannii, illustrating the importance of polymicrobial nature in investigating S. aureus pathogenesis. The infection-type-specific genes likely serve as potential therapeutic targets to control S. aureus infections, either in mono- or co-infection situation, providing novel insights into the development of antimicrobial regimens to control co-infections.

Emergence of a Carbapenem-Resistant Klebsiella pneumoniae Isolate Co-harbouring Dual bla NDM- 6 -Carrying Plasmids in China.

The widespread emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) with limited therapeutic options has become a global concern. In this study, a K. pneumoniae strain called KP2e was recovered from a human case of fatal septic shock in a Chinese hospital. Polymerase chain reaction and sequencing, antimicrobial susceptibility testing, conjugation experiments, S1 nuclease-pulsed field gel electrophoresis/southern blot, whole genome sequencing and comparative genomics were performed to investigate the phenotypic and molecular characteristics of this isolate. KP2e possessed the NDM-6-encoding gene and exhibited resistance to almost all ß-lactams except for monobactam. This strain belonged to sequence type 4024, the complete genome of which was composed of one chromosome and three plasmids. Furthermore, bla NDM-6 coexisted on two self-transmissible plasmids, which were assigned to types IncFIB and IncN. A structure of IS26-composite transposon capturing an identical Tn125 remnant (ΔISAba125-bla NDM-6 -ble MBL -trpF-dsbC-cutA-groES-ΔgroEL) was identified in the two plasmids, and this conserved bla NDM -surrounding genetic context was similar to that of few IncN plasmids found in other regions of China. Our research appears to be the first description of a clinical strain that emerged co-harbouring dual bla NDM -carrying plasmids, and the first report of NDM-6-positive CRKP in China. These findings demonstrated that IncN is a key medium in the evolution and expanding dissemination of bla NDM genes among various species, which indicates that close monitoring and rapid detection of bla NDM -harbouring plasmids is necessary.

Late-Onset Acute Kidney Injury is a Poor Prognostic Sign for Severe Burn Patients.

Background: Acute kidney injury (AKI) is a morbid complication and the main cause of multiple organ failure and death in severely burned patients. The objective of this study was to explore epidemiology, risk factors, and outcomes of AKI for severely burned patients. Methods: This retrospective study was performed with prospectively collected data of severely burned patients from the Institute of Burn Research in Southwest Hospital during 2011-2017. AKI was diagnosed according to Kidney Disease Improving Global Outcomes (KDIGO) criteria (2012), and it was divided into early and late AKIs depending on its onset time (within the first 3 days or >3 days post burn). The baseline characteristics, clinical data, and outcomes of the three groups (early AKI, late AKI and non-AKI) were compared using logistic regression analysis. Mortality predictors of patients with AKI were assessed. Results: A total of 637 adult patients were included in analysis. The incidence of AKI was 36.9% (early AKI 29.4%, late AKI 10.0%). Multiple logistic regression analysis revealed that age, gender, total burn surface area (TBSA), full-thickness burns of TBSA, chronic comorbidities (hypertension or/and diabetes), hypovolemic shock of early burn, and tracheotomy were independent risk factors for both early and late AKIs. However, sepsis was only an independent risk factor for late AKI. Decompression escharotomy was a protective factor for both AKIs. The mortality of patients with AKI was 32.3% (early AKI 25.7%, late AKI 56.3%), and that of patients without AKI was 2.5%. AKI was independently associated with obviously increased mortality of severely burned patients [early AKI, OR = 12.98 (6.08-27.72); late AKI, OR = 34.02 (15.69-73.75)]. Compared with patients with early AKI, patients with late AKI had higher 28-day mortality (34.9% vs. 19.4%, p = 0.007), 90-day mortality (57.1% vs. 27.4%, p < 0.0001). Conclusions: AKI remains prevalent and is associated with high mortality in severely burned patients. Late-onset acute kidney injury had greater severity and worse prognosis.

Dynamics of Diversity and Abundance of Sulfonamide Resistant Bacteria in a Silt Loam Soil Fertilized by Compost.

Although composting is effective in deactivating antibiotic substances in manure, the influence of compost fertilization on the occurrence and dissemination of antibiotic resistance in arable soils remains to be controversial. Herein, the abundance and diversity of two sulfonamide resistance genes (sul1 and sul2) in soil fertilized by compost spiked with two concentrations of sulfadiazine (1 and 10 mg kg-1) were studied intensively by qPCR and high throughput sequencing based on a two-month microcosm experiment. The concentration of sulfadiazine decreased rapidly after spiking from 25% at Day 1 to less than 2.7% at Day 60. Relative abundance of both sul1 and sul2 were significantly higher in soil amended with compost than the non-amended control at Day 1 and slightly decreased with incubation time except for sul2 in the S10 treatment. Soil bacterial communities were transiently shifted by compost fertilization regardless of the presence of sulfadiazine. Relative abundance of genera in three hubs positively interlinked with sul1 and sul2 were significantly higher in compost treated soil than the control at Day 1, 7 and 21, but not at Day 60. High throughput sequencing analyses revealed that most detected (>67% in relative abundance) sul1 and sul2 genotypes sharing >99% similarity with those found in gammaproteobacterial pathogens frequently were commonly present in compost and soil. These results indicated that compost fertilization might increase the abundance rather than diversity of sulfadiazine-resistant populations in soil, which may be facilitated by the presence of sulfadiazine.

Different Infection Profiles and Antimicrobial Resistance Patterns Between Burn ICU and Common Wards.

Infection is the leading cause of complications and deaths after burns. However, the difference in infection patterns between the burn intensive care unit (BICU) and burn common wards (BCW) have not been clearly investigated. The present study aimed to compare the infection profile, antimicrobial resistance, and their changing patterns in burn patients in BICU and BCW. Clinical samples were analyzed between January 1, 2011, and December 31, 2019, in the Institute of Burn Research in Southwest China. The patient information, pathogen distribution, sources, and antimicrobial resistance were retrospectively collected. A total of 3457 and 4219 strains were detected in BICU and BCW, respectively. Wound secretions accounted for 86.6% and 44.9% in BCW and BICU, respectively. Compared with samples in BCW, samples in BICU had more fungi (11.8% vs. 8.1%), more Gram-negative bacteria (60.0% vs. 50.8%), and less Gram-positive bacteria (28.2% vs. 41.1%). Acinetobacter baumannii were the most common pathogen in BICU, compared with Staphylococcus aureus in BCW. S. aureus was the most frequent pathogen in wound secretions and tissues from both BICU and BCW. However, A. baumannii were the first in blood, sputum, and catheter samples from BICU. Overall, the multidrug-resistance (MDR) rate was higher in BICU than in BCW. However, the gap between BICU and BCW gradually shortened from 2011 to 2019. The prevalence of MDR A. baumannii and Klebsiella pneumonia significantly increased, especially in BCW. Furthermore, Carbapenem resistance among K. pneumoniae significantly increased in BICU (4.5% in 2011 vs. 40% in 2019) and BCW (0 in 2011 vs. 40% in 2019). However, the percentage of MDR P. aeruginosa sharply dropped from 85.7% to 24.5% in BICU. The incidence of MRSA was significantly higher in BICU than in BCW (94.2% vs. 71.0%) and stayed at a high level in BICU (89.5% to 96.3%). C. tropicalis and C. albicans were the two most frequent fungi. No resistance to Amphotericin B was detected. Our study shows that the infection profile is different between BICU and BCW, and multidrug resistance is more serious in BICU than BCW. Therefore, different infection-control strategies should be emphasized in different burn populations.

Safety and Efficacy of a Phage, kpssk3, in an in vivo Model of Carbapenem-Resistant Hypermucoviscous Klebsiella pneumoniae Bacteremia.

Antimicrobial resistance (AMR) is one of the most significant threats to global public health. As antibiotic failure is increasing, phages are gradually becoming important agents in the post-antibiotic era. In this study, the therapeutic effects and safety of kpssk3, a previously isolated phage infecting carbapenem-resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP), were evaluated in a mouse model of systemic CR-HMKP infection. The therapeutic efficacy experiment showed that intraperitoneal injection with a single dose of phage kpssk3 (1 × 107 PFU/mouse) 3 h post infection protected 100% of BALB/c mice against bacteremia induced by intraperitoneal challenge with a 2 × LD100 dose of NY03, a CR-HMKP clinical isolate. In addition, mice were treated with antibiotics from three classes (polymyxin B, tigecycline, and ceftazidime/avibactam plus aztreonam), and the 7 days survival rates of the treated mice were 20, 20, and 90%, respectively. The safety test consisted of 2 parts: determining the cytotoxicity of kpssk3 and evaluating the short- and long-term impacts of phage therapy on the mouse gut microbiota. Phage kpssk3 was shown to not be cytotoxic to mammalian cells in vitro or in vivo. Fecal samples were collected from the phage-treated mice at 3 time points before (0 day) and after (3 and 10 days) phage therapy to study the change in the gut microbiome via high-throughput 16S rDNA sequence analysis, which revealed no notable alterations in the gut microbiota except for decreases in the Chao1 and ACE indexes.

Procalcitonin kinetics early after severe burn injury and its value in diagnosis of sepsis.

PURPOSE: To investigate the clinical significance of procalcitonin (PCT) kinetics early after burn and the perioperative period, and to assess its diagnostic performance for sepsis in major burn patients. METHODS: This retrospective study on major burn patents (≥40% total body surface area) admitted from 2014 to 2019 was conducted in Southwest Hospital, Chongqing, China. A total of 321 patients were included. The kinetics of PCT was analyzed during the 1st week after burn, the perioperative period, and at the onset of clinical suspected sepsis. RESULTS: Serum PCT concentration rose immediately after burn injury. Factors associated with increased PCT level in the 1st week after burn include greater burn area (>70% TBSA) and lower age (≤14 years). Correlations between PCT kinetics after burn and the risk of early development of sepsis and mortality were observed. At the onset of sepsis, serum PCT increased significantly compared to its basal level in the 48 h before diagnosis. The area under the receiver operating characteristics curve of PCT concentration and its kinetic changes was 0.788 and 0.826, respectively. PCT kinetics showed better accuracy than PCT concentration in discrimination of Gram-positive sepsis. The optimal diagnostic thresholds for PCT concentration and its kinetics were 1.41 ng/mL, and a 1.34-fold elevation compared to the baseline level. CONCLUSIONS: PCT kinetics in the early stage after burn was a prognostic factor for sepsis and mortality among major burn patients. Serum PCT levels could be a diagnostic biomarker for sepsis in major burn patients.

Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. METHODS: In this study, a 15-µL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the bla OXA-51 gene by PCR, qPCR and 15 µL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 µL-RPA data for detecting the carbapenem-resistance gene bla OXA-23 were comparatively assessed. RESULTS: The detection limit of the bla OXA-51 gene by 15 µL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 µL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 µL RPA data for detecting bla OXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. CONCLUSION: We demonstrated that the new 15 µL RPA assay for detecting bla OXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

Characterization and genome sequencing of a novel T7-like lytic phage, kpssk3, infecting carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has spread globally and emerged as an urgent public health threat. Bacteriophages are considered an effective weapon against multidrug-resistant pathogens. In this study, we report a novel lytic phage, kpssk3, which is able to lyse CRKP and degrade exopolysaccharide (EPS). The morphological characteristics of kpssk3 observed by transmission electron microscopy, including a polyhedral head and a short tail, indicate that it belongs to the family Podoviridae. A one-step growth curve revealed that kpssk3 has a latent period of 10 min and a burst size of 200 plaque-forming units (pfu) per cell. kpssk3 was able to lyse 25 out of 27 (92.59%) clinically isolated CRKP strains, and it also exhibited high stability to changes in temperature and pH. kpssk3 has a linear dsDNA genome of 40,539 bp with 52.80% G+C content and 42 putative open reading frames (ORFs). No antibiotic resistance genes, virulence factors, or integrases were identified in the genome. Based on bioinformatic analysis, the tail fiber protein of phage kpssk3 was speculated to possess depolymerase activity towards EPS. By comparative genomics and phylogenetic analysis, it was determined that kpssk3 is a new T7-like virus and belongs to the subfamily Autographivirinae. The characterization and genomic analysis of kpssk3 will promote our understanding of phage biology and diversity and provide a potential strategy for controlling CRKP infection.

Lytic Bacteriophage Screening Strategies for Multidrug-Resistant Bloodstream Infections in a Burn Intensive Care Unit.

BACKGROUND Increasing antibiotic resistance and multidrug resistance (MDR) in patients with bloodstream infection (BSI) has resulted in treatment using bacteriophage. This study aimed to identify Gram-negative bacilli and Gram-positive cocci and antibiotic resistance in patients with BSI in a burn intensive care unit (BICU). The environment, including sewage systems, were investigated for the presence of lytic bacteriophage. MATERIAL AND METHODS Between January 2011 to December 2017, 486 patients with BSI were admitted to the BICU. Blood culture identified the main infectious organisms. Bacterial screening tests for antibiotic resistance included the D test and the modified Hodge test (MHT). Lytic bacteriophage was isolated from the environment. RESULTS In 486 patients with BSI, the main causative organisms were Gram-negative bacilli (64.6%), Gram-positive cocci (27.7%), and fungi (7.7%). The main pathogenic organisms that showed multidrug resistance (MDR) were Acinetobacter baumannii (26.0%), Staphylococcus aureus (16.8%), and Pseudomonas aeruginosa (14.2%). Bacteriophage was mainly isolated from Gram-negative bacilli. Screening of hospital and residential sewage systems identified increased levels of bacteriophage in hospital sewage. CONCLUSIONS The causative organisms of BSI and the presence of MDR in a hospital BICU were not typical, which supports the need for routine bacterial monitoring. Hospital sewage provides a potential source of bacteriophage for the treatment of MDR pathogenic bacteria.

Global Transcriptomic Analysis of the Interactions between Phage φAbp1 and Extensively Drug-Resistant Acinetobacter baumannii.

Acinetobacter baumannii is a growing threat, although lytic bacteriophages have been shown to effectively kill A. baumannii. However, the interaction between the host and the phage has not been fully studied. We demonstrate the global profile of transcriptional changes in extensively drug-resistant A. baumannii AB1 and the interaction with phage φAbp1 through RNA sequencing (RNA-seq) and bioinformatic analysis. Only 15.6% (600/3,838) of the genes of the infected host were determined to be differentially expressed genes (DEGs), indicating that only a small part of the bacterial resources was needed for φAbp1 propagation. Contrary to previous similar studies, more upregulated rather than downregulated DEGs were detected. Specifically, φAbp1 infection caused the most extensive impact on host gene expression at 10 min, which was related to the intracellular accumulation phase of virus multiplication. Based on the gene coexpression network, a middle gene (gp34, encoding phage-associated RNA polymerase) showed a negative interaction with numerous host ribosome protein genes. In addition, the gene expression of bacterial virulence/resistance factors was proven to change significantly. This work provides new insights into the interactions of φAbp1 and its host, which contributes to the further understanding of phage therapy, and provides another reference for antibacterial agents. IMPORTANCE Previous research has reported the transcriptomic phage-host interactions in Escherichia coli and Pseudomonas aeruginosa, leading to the detailed discovery of transcriptomic regulations and predictions of specific gene functions. However, a direct relationship between A. baumannii and its phage has not been previously reported, although A. baumannii is becoming a rigorous drug-resistant threat. We analyzed transcriptomic changes after φAbp1 infected its host, extensively drug-resistant (XDR) A. baumannii AB1, and found defense-like responses of the host, step-by-step control by the invader, elaborate interactions between host and phage, and elevated drug resistance gene expressions of AB1 after phage infection. These findings suggest the detailed interactions of A. baumannii and its phage, which may provide both encouraging suggestions for drug design and advice for the clinical use of vital phage particles.

Succinylated casein-coated peptide-mesoporous silica nanoparticles as an antibiotic against intestinal bacterial infection.

Increasing drug resistance necessitates the discovery of novel bactericides. Human defensin (HD) peptides can eliminate resistant bacteria and are promising candidates for next-generation antibiotics. T7E21R-HD5 is a potent bactericide designed by site mutations at enteric HD5. To facilitate the development of T7E21R-HD5 into an intestinal antibiotic, we employed a mesoporous silica nanoparticle (MSN) as the peptide carrier. Despite its ineffectiveness at killing bacteria, the MSN intensified the outer membrane penetration and inner membrane permeabilization abilities of T7E21R-HD5 and thus enhanced its antibacterial action against multidrug resistant (MDR) E. coli, which broadened the role of MSNs in drug delivery. For the reduction in T7E21R-HD5 losses in the stomach, we further modified MSN@T7E21R-HD5 with succinylated casein (SCN), a milk protein that can be specifically degraded by intestinal protease. SCN coating decreased T7E21R-HD5 release from the MSNs, especially in a highly acidic environment. The controlled release of MSN@T7E21R-HD5 from SCN encapsulation was confirmed in the presence of trypsin. MSN@T7E21R-HD5@SCN was nontoxic to host cells, and it was capable of inactivating MDR E. coli in vivo and alleviating intestinal inflammation by suppressing the production of inflammatory factors TNF-α, IL-1ß, and MMP-9. This study provides a peptide-based nanobiotic with efficacy to combat intestinal infection, especially against drug-resistant bacteria. The biocompatible and readily prepared MSN/SCN delivery system may benefit further intestinal antibiotic design and promote the drug transformation of additional enterogenic functional molecules.