Antimicrobial Resistance and Molecular Characterization of Escherichia coli from Healthy Chickens in Shandong, China from 2009 to 2014.

Antimicrobial resistance (AMR) is a great threat to animal and public health. Here, we conducted a surveillance of Escherichia coli isolated from healthy chickens during 2009-2014 to identify the characteristics of AMR. A total of 351 (95.64%) E. coli isolates were obtained from 367 healthy chicken fecal samples collected from 6 farms located in Shandong Province, China. The susceptibility to 10 antimicrobials, the prevalence of antibiotic resistance genes (ARGs), phylogenetic clustering, and multilocus sequence typing were evaluated. The isolates exhibited high resistant rates (>95%) to ampicillin, cefotaxime, ciprofloxacin, ceftiofur, and enrofloxacin. The most prevalent ARGs were blaCTX-M (36.36%), aac(6')-Ib-cr (30.79%), qnrS (29.62%), oqxAB (27%), mcr-1 (15.83%), blaTEM (9.09%), qnrC (3.52%), qnrD (0.88%), and qepA (0.29%). Phylogenetic clustering analysis indicated that the most prevalent group was group D (37.89%), followed by group B1 (34.76%), A (24.22%), and B2 (3.13%). Fifty-seven sequence types (STs) were identified among the 124 blaCTX-M-positive strains, and the dominant STs were ST354 (13.71%), ST117 (5.65%), ST155, ST2309, and ST2505 (4.84% each). There was a significant association between 17 pairs of AMR phenotypes, 14 pairs of ARGs, and 11 pairs of AMR-ARGs. The strongest association was found between ST602 and qnrC (odds ratios: 22.2). This study implied that E. coli isolated from healthy chickens could potentially serve as a reservoir of AMR and ARGs, and significant associations exist among AMR, ARGs, phylogenetic groups, and STs. Our study highlighted the need for routine surveillance of AMR in healthy chickens, and promoting appropriate antibiotic use and implementing regular monitoring of resistance in broilers are crucial for fostering the development of the poultry industry and safeguarding public health.

Disarm The Bacteria: What Temperate Phages Can Do.

In the field of phage applications and clinical treatment, virulent phages have been in the spotlight whereas temperate phages received, relatively speaking, less attention. The fact that temperate phages often carry virulent or drug-resistant genes is a constant concern and drawback in temperate phage applications. However, temperate phages also play a role in bacterial regulation. This review elucidates the biological properties of temperate phages based on their life cycle and introduces the latest work on temperate phage applications, such as on host virulence reduction, biofilm degradation, genetic engineering and phage display. The versatile use of temperate phages coupled with their inherent properties, such as economy, ready accessibility, wide variety and host specificity, make temperate phages a solid candidate in tackling bacterial infections.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Phage Engineering for Targeted Multidrug-Resistant Escherichia coli.

The lytic bacteriophages have potential application value in the treatment of bacterial infections. However, the narrow host spectrum of these phages limits their range of clinical application. Here, we demonstrate the use of scarless Cas9-assisted recombination (no-SCAR) gene-editing technology to regulate phage-host range. We used phage PHB20 as the scaffold to create agents targeting different multidrug-resistant Escherichia coli by replacing its phage tail fiber gene (ORF40). The engineered phages were polyvalent and capable of infecting both the original host bacteria and new targets. Phage-tail fiber genes can be amplified by PCR to construct a recombinant phage PHB20 library that can deal with multidrug-resistant bacteria in the future. Our results provide a better understanding of phage-host interactions, and we describe new anti-bacterial editing methods.

A Multifunctional Light-Driven Swimming Soft Robot for Various Application Scenarios.

The locomotor behavior of creatures in nature can bring a lot of inspiration for the fabrication of soft actuators. In this paper, we fabricated a bionic light-driven swimming soft robot that can perform grasping of tiny objects and achieve the task of object transfer. By adding carbon nanotubes (CNTs), the temperature-sensitive hydrogels can be endowed with light-responsive properties. The fabricated composite hydrogel structure can control the contraction and expansion of volume by light, which is similar to the contraction and diastole behavior of muscles. The oscillation of the fish tail and the grasping action of the normally closed micromanipulator can be achieved by the control of the irradiation of the xenon light source. The bending of the bionic arm can be controlled by the irradiation of a near-infrared (NIR) laser, which transforms the spatial position and posture of the micromanipulator. The proposed scheme is feasible for miniaturized fabrication and application of flexible actuators. This work provides some important insights for the study of light-driven microrobots and light-driven flexible actuators.

A Novel Tail-Associated O91-Specific Polysaccharide Depolymerase from a Podophage Reveals Lytic Efficacy of Shiga Toxin-Producing Escherichia coli.

Shiga toxin-producing Escherichia coli (STEC) strains are important zoonotic foodborne pathogens, causing diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. However, antibiotic treatment of STEC infection is associated with an increased risk of HUS. Therefore, there is an urgent need for early and effective therapeutic strategies. Here, we isolated lytic T7-like STEC phage PHB19 and identified a novel O91-specific polysaccharide depolymerase (Dep6) in the C terminus of the PHB19 tailspike protein. Dep6 exhibited strong hydrolase activity across wide ranges of pH (pH 4 to 8) and temperature (20 to 60°C) and degraded polysaccharides on the surface of STEC strain HB10. In addition, both Dep6 and PHB19 degraded biofilms formed by STEC strain HB10. In a mouse STEC infection model, delayed Dep6 treatment (3 h postinfection) resulted in only 33% survival, compared with 83% survival when mice were treated simultaneously with infection. In comparison, pretreatment with Dep6 led to 100% survival compared with that of the control group. Surprisingly, a single PHB19 treatment resulted in 100% survival in all three treatment protocols. Moreover, a significant reduction in the levels of proinflammatory cytokines was observed at 24 h postinfection in Dep6- or PHB19-treated mice. These results demonstrated that Dep6 or PHB19 might be used as a potential therapeutic agent to prevent STEC infection.IMPORTANCE Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen worldwide. The Shiga-like toxin causes diarrhea, hemorrhagic colitis, and life-threatening hemolytic uremic syndrome (HUS) in humans. Although antibiotic therapy is still used for STEC infections, this approach may increase the risk of HUS. Phages or phage-derived depolymerases have been used to treat bacterial infections in animals and humans, as in the case of the "San Diego patient" treated with a phage cocktail. Here, we showed that phage PHB19 and its O91-specific polysaccharide depolymerase Dep6 degraded STEC biofilms and stripped the lipopolysaccharide (LPS) from STEC strain HB10, which was subsequently killed by serum complement in vitro In a mouse model, PHB19 and Dep6 protected against STEC infection and caused a significant reduction in the levels of proinflammatory cytokines. This study reports the use of an O91-specific polysaccharide depolymerase for the treatment of STEC infection in mice.

Recent advances in microfluidic technologies for separation of biological cells.

Cell separation has always been a key topic in academic research, especially in the fields of medicine and biology, due to its significance in diagnosis and treatment. Accurate, high-throughput and non-invasive separation of individual cells is key to driving the development of biomedicine and cellular biology. In recent years, a series of researches on the use of microfluidic technologies for cell separation have been conducted to solve bio-related problems. Hence, we present here a comprehensive review on the recent developments of microfluidic technologies for cell separation. In this review, we discuss several cell separation methods, mainly including: physical and biochemical method, their working principles as well as their practical applications. We also analyze the advantages and disadvantages of each method in detail. In addition, the current challenges and future prospects of microfluidic-based cell separation were discussed.

Characterisation of a newly detected bacteriophage infecting Bordetella bronchiseptica in swine.

A novel virulent bacteriophage, vB_BbrM_PHB04, infecting Bordetella bronchiseptica was isolated from wastewater collected at a swine farm in China. Phage vB_BbrM_PHB04 exhibited growth over a wide range of temperature and pH conditions and showed different efficiency of plating values and lytic spectra within the same strains at 25 °C and 37 °C. High-throughput sequencing revealed that vB_BbrM_PHB04 has a linear double-stranded DNA genome with 124 putative open reading frames. Overall, the genome of vB_BbrM_PHB04 showed very low similarity (the highest nucleotide identity 82%, 1% coverage) to other phage sequences in the GenBank database. Phylogenetic analysis indicated that vB_BbrM_PHB04 is a new member of the family Myoviridae. In addition, polymerase chain reaction-based detection of phage genes in phage-resistant B. bronchiseptica variants revealed no evidence of lysogenic activity of phage vB_BbrM_PHB04.

Complete genome sequence of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli.

The complete genome of the novel phage vB_EcoS_PHB17, which infects Shiga-toxin-producing Escherichia coli, was sequenced, revealing a linear double-stranded DNA genome of 48,939 bp with 46% GC content and protruding 150-bp 5' cohesive termini. The genome contained 85 open reading frames, 28 of which were annotated with known functions. No tRNA-encoding genes were detected. Phylogenetic analysis suggested that phage PHB17 is a novel phage of family Siphoviridae.

Isolation and characterization of a novel temperate bacteriophage from gut-associated Escherichia within black soldier fly larvae (Hermetia illucens L. [Diptera: Stratiomyidae]).

To gain insight into the presence and nature of prophages in the black soldier fly (BSF; Hermetia illucens L. [Diptera: Stratiomyidae]) gut, we isolated and characterized a novel, temperate Escherichia bacteriophage designated vB_EcoS_PHB10 (PHB10). Electron microscopy analysis revealed that phage PHB10 has a long, flexible, non-contractile tail and belongs to the family Siphoviridae. The phage was found to be stable over a wide range of temperatures (4-37 °C) and pH values (pH 5-9), and it lysed two out of 13 Escherichia strains tested. The genome of PHB10 contains genes encoding a putative transcriptional regulator and an integrase, and it shows a high degree of similarity to a region of the Enterobacter cloacae MBRL1077 genome. Induction experiments revealed that phage PHB10 could be induced by different gut substrates, suggesting that diet might be a potential regulator of lytic/lysogenic switches in commensal lysogens.

A capsule/lipopolysaccharide/MLST genotype D/L6/ST11 of Pasteurella multocida is likely to be strongly associated with swine respiratory disease in China.

Pasteurella multocida is a leading cause of respiratory disease in pigs worldwide. In this study, we determined the genetic characteristics of 115 P. multocida isolates from the lungs of pigs with respiratory disease in China in 2015 using capsular typing, lipopolysaccharide (LPS) genotyping, and virulence genotyping based on the detection of virulence-associated genes. The results showed that the isolates belonged to three capsular types: A (49.6%), D (46.1%), and nontypable (4.3%); and two LPS genotypes: L3 (22.6%) and L6 (77.4%). When combining the capsular types with the LPS genotypes, a genotype group D: L6 (46.1%) was the most prevalent among the strains. Among the 23 virulence-associated genes detected in this study, a small number of them displayed a certain level of "genotype-preference". We found that pfhA, hgbA, and hgbB had a close association with P. multocida LPS genotypes, while tadD was more associated with P. multocida capsular types. In addition, multilocus sequence typing (MLST) on 40 P. multocida isolates identified four sequence types: ST3, ST10, ST11, and ST16, and the distribution of ST11 was significantly higher than the other MLST genotypes. Interestingly, all of the ST11 isolates detected in this study were genotype D: L6 strains and they were 100% positive for hgbB. Our data suggest that a capsule/LPS/MLST genotype D/L6/ST11 is likely to be strongly associated with respiratory clinical manifestation of the disease in pigs.

Synthesis, Crystal Structure, and Optical Gap of Two-Dimensional Halide Solid Solutions CsPb2(Cl1- xBr x)5.

Exploring new perovskite-related solid-state materials and the investigating composition-dependent structural and physical properties are highly important for advanced functional material development. Herein, we present the successful hydrothermal synthesis of tetragonal CsPb2Cl5 and the anion-exchange phase formation of CsPb2(Cl1- xBr x)5 ( x = 0-1) solid solutions. The CsPb2(Cl1- xBr x)5 crystal structures, which crystallize in the tetragonal system, space group I4/ mcm, with parameters similar to those of CsPb2Cl5, have been determined by Rietveld analysis. The optical band gap was obtained by UV-vis spectroscopy, and the band structure was further calculated by the full-potential method within the generalized gradient approximation. It was revealed that the band gap in CsPb2(Cl1- xBr x)5 solid solutions can be tuned over the range of 4.5-3.8 eV by anion substitution.

Three Salmonella enterica serovar Enteritidis bacteriophages from the Siphoviridae family are promising candidates for phage therapy.

Salmonella is a common and widely distributed foodborne pathogen that is frequently implicated in gastrointestinal infections. The emergence and spread of Salmonella strains resistant to multiple antibiotics poses a significant health threat, highlighting the urgent need for early and effective therapeutic strategies. We isolated a total of 32 phages from water samples and anal swabs from pigs. Of these, three phages that produced large, clear plaques were selected for further study using the following methods: electron microscopy, analysis of the life cycle parameters, genetic analysis, inhibition of bacterial growth, and activity against biofilms. The three Salmonella phages (vB_SenS_CSP01, vB_SenS_PHB06, and vB_SenS_PHB07) were assigned to the family Siphoviridae on the basis of their morphology. All showed polyvalent infectivity, and individual phages or phage cocktails could inhibit the growth of host Salmonella enterica serovar Enteritidis strains or reduce biofilm formation by Salmonella enterica serovar Typhimurium. In summary, these three phages merit further research as biocontrol agents for Salmonella infection.

Complete Genome Sequence of a Novel T7-Like Bacteriophage from a Pasteurella multocida Capsular Type A Isolate.

A novel virulent bacteriophage, vB_PmuP_PHB02 (phage PHB02), infecting Pasteurella multocida capsular type A strains, was isolated from wastewater from a swine farm in China. Phage PHB02 has a linear double-stranded DNA genome consisting of 38,670 base pairs (bp), with a G+C content of 40.8% and a 127-bp terminal redundancy. Forty-eight putative open reading frames were identified, and no transfer RNA-encoding genes were detected. The morphology and genomic structure of phage PHB02 resemble those of T7-like phages belonging to the family Podoviridae, of the order Caudovirales. Phage PHB02 was stable over a wide range of temperatures (4-50 °C) and pH values (5.0-9.0), and lysed 30 of the 31 capsular-type-A P. multocida strains tested. Phage PHB02 had no effect on other bacterial species or on P. multocida strains belonging to capsular types D or F.

Light-driven soft microrobots based on hydrogels and LCEs: development and prospects.

In the daily life of mankind, microrobots can respond to stimulations received and perform different functions, which can be used to complete repetitive or dangerous tasks. Magnetic driving works well in robots that are tens or hundreds of microns in size, but there are big challenges in driving microrobots that are just a few microns in size. Therefore, it is impossible to guarantee the precise drive of microrobots to perform tasks. Acoustic driven micro-nano robot can achieve non-invasive and on-demand movement, and the drive has good biological compatibility, but the drive mode has low resolution and requires expensive experimental equipment. Light-driven robots move by converting light energy into other forms of energy. Light is a renewable, powerful energy source that can be used to transmit energy. Due to the gradual maturity of beam modulation and optical microscope technology, the application of light-driven microrobots has gradually become widespread. Light as a kind of electromagnetic wave, we can change the energy of light by controlling the wavelength and intensity of light. Therefore, the light-driven robot has the advantages of programmable, wireless, high resolution and accurate spatio-temporal control. According to the types of robots, light-driven robots are subdivided into three categories, namely light-driven soft microrobots, photochemical microrobots and 3D printed hard polymer microrobots. In this paper, the driving materials, driving mechanisms and application scenarios of light-driven soft microrobots are reviewed, and their advantages and limitations are discussed. Finally, we prospected the field, pointed out the challenges faced by light-driven soft micro robots and proposed corresponding solutions.

Characterization, taxonomic classification, and genomic analysis of two newly isolated bacteriophages with potential to infect Escherichia coli.

Escherichia coli is a pathogenic bacterium that is widely distributed and can lead to serious illnesses in both humans and animals. As there is rising incidence of multidrug resistance among these bacteria, it has become imperative to discover alternative therapies beyond antibiotics to effectively treat such infections. Bacteriophage (phage) therapy has the potential to treat infections caused by E. coli, as phages contain enzymes that can cause lysis or destruction of bacterial cells. Simultaneously, the easy accessibility and cost-effectiveness of next-generation sequencing technologies have led to the accumulation of a vast amount of phage sequence data. Here, phages IME177 and IME267 were isolated from sewage water of a hospital in China. Modern phylogenetic approaches and key findings from the genomic analysis revealed that phages IME177 and IME267 are classified as members of the Kayfunavirus genus, Autographiviridae family, and a newly proposed Suseptimavirus genus under subfamily Gordonclarkvirinae, respectively. Further, the Kuravirus genus reshaped into three different genera: Kuravirus, Nieuwekanaalvirus, and Suspeptimavirus, which are classified together under a higher taxonomic rank (subfamily) named Gordonclarkvirinae. No genes related to virulence were detected in the genomes of the phages IME177 and IME267. Both phages exhibited a high degree of resilience to a wide range of conditions, including pH, temperature, exposure to chloroform, and UV radiation. Phages IME177 and IME267 are promising biological agents that can infect E. coli, making them suitable candidates for use in phage therapies.IMPORTANCEBiological and taxonomic characterization of phages is essential for facilitating the development of effective strategies for phage therapy and disease control. Escherichia coli phages are incredibly diverse, and their isolation and classification help us understand the scope and nature of this diversity. By identifying new phages and grouping them into families, we can better understand the genetic and structural variations between phages and how they affect their infectivity and interactions with bacteria. Overall, the isolation and classification of E. coli phages have broad implications for both basic and applied research, clinical practice, and public health.

Genome sequence of Shiga toxin-producing Escherichia coli jumbo bacteriophage vB_EcoM_JNE01.

Bacteriophage vB_EcoM_JNE01 was isolated from chicken farm sewage using Escherichia coli O157:H7 as the host bacteria. The total length of the vB_EcoM_JNE01 genome is 355,583 bp, with 584 open reading frames and 36% G+C content. It shares an 80% nucleotide identity with 59% query coverage with the bacteriophage PBECO4 (NC_027364).

Characterization, genome analysis, and therapeutic evaluation of a novel Salmonella phage vB_SalS_JNS02: a candidate bacteriophage for phage therapy.

Phage therapy is gaining momentum as an alternative to antibiotics in the treatment of salmonellosis caused by Salmonella. In this study, a novel Salmonella phage, vB_SalS_JNS02, was isolated successfully from poultry farms in Shandong, China. The biological characteristics of vB_SalS_JNS02 were analysed, which revealed a short latent period of approximately 10 min and a burst size of 110 PFU/cell. Moreover, vB_SalS_JNS02 exhibited remarkable stability across a wide pH range (pH 3-12) and temperatures ranging from 30 to 80°C. Genome sequencing analysis provided valuable insights into the genetic composition of vB_SalS_JNS02, which consists of a double-stranded DNA genome that spans 42,450 base pairs and has a G + C content of 49.4%. Of significant importance, the genomic sequence of vB_SalS_JNS02 did not contain any genes related to lysogenicity, virulence, or antibiotic resistance. The phage's efficacy was evaluated in a larval challenge study. Treatment with the phage resulted in increased survival of Galleria mellonella larvae (100, 70, and 85%) (MOI 0.1) in the prophylactic treatment, co-infection treatment, and remedial treatment experiments, respectively. Another in vivo experiment investigated the potential application of the phage in broiler chickens and revealed that a single oral dose of vB_SalS_JNS02 (108 PFU/mL, 100 µL/chick) administered 3 h after S. enteritidis oral administration provided effective protection. The introduction of bacteriophage not only enhances the production of secretory immunoglobulin A (sIgA), but also induces alterations in the composition of the gut microbial community. Phage therapy increases the relative abundance of beneficial bacteria, which helps to maintain intestinal barrier homeostasis. However, it is unable to fully restore the disrupted intestinal microbiome caused by S. enteritidis infection. Importantly, no significant adverse effects were observed in the animal subjects following oral administration of the phage, and our findings highlight vB_SalS_JNS02 is a hopeful candidate as a promising tool to target Salmonella infections in poultry.

Genomic analysis and experimental pathogenic characterization of Riemerella anatipestifer isolates from chickens in China.

Waterfowl have a high likelihood of being infected with Riemerella anatipestifer. Although the pathogen is found in domestic ducks, turkeys, geese, and wild birds, there is little information available about the consequences of infection during egg laying and hatching in chickens. Here, we present the first report of a novel sequence type of R. anatipestifer S63 isolated from chickens in China. On the basis of pan-genome analysis, we showed S63's genome occupies a distinct branch with other R. anatipestifer isolates from other hosts. Galleria mellonella larval tests indicated that S63 is less virulent than R. anatipestifer Ra36 isolated from ducks. Ducks and hens are susceptible to S63 infection. There is no mortality rate for chickens or ducks, but adult chickens experience neurological symptoms that reduce egg production and hatching rates. In chickens, S63 might be passed vertically from parents to offspring, resulting in "jelly-like" lifeless embryos. Using quantitative PCR, S63 was detected in the brain, liver, reproductive organs, and embryos. As far as we know, this is the first report of R. anatipestifer in hens, a disease that can reduce egg productivity, lower hatching rates, and produce jelly-like lifeless embryos, and the first report to raise the possibility that hens can be infected by roosters via semen.

Toxin gene detection and antibiotic resistance of Clostridium perfringens from aquatic sources.

Clostridium perfringens is a zoonotic opportunistic pathogen that produces toxins that can cause necrotic enteritis and even "sudden death disease". This bacterium is widely distributed in the intestines of livestock and human, but there are few reports of distribution in aquatic animals (Hafeez et al., 2022). In order to explore the isolation rate of C. perfringens and the toxin genes they carry, 141 aquatic samples, including clams (Ruditapes philippinarum), oysters (Ostreidae), and mud snails (Bullacta exerata Philippi), were collected from the coastal areas of Shandong Province, China. C. perfringens strains were tested for cpa, cpb, etx, iap, cpb2, cpe, netB, and tpeL genes. 45 clam samples were boiled at 100 °C for 5 min before bacteria isolation. 80 strains were isolated from 141 samples with the positive rate being 57 %.And the positive rates of cooked clams was 87 % which was higher than the average. In detection of 8 toxin genes, all strains tested cpa positive, 3 strains netB positive, and 2 cpb and cpe, respectively. 64 strains were selected to analyze the antibiotic resistance phenotype of 10 antibiotics. The average antibiotic resistance rates of the strains to tetracycline, clindamycin, and ampicillin were 45 %, 20 %, and 16 % respectively, and the MIC of 4 strains to clindamycin was ≥128 µg/mL. A high isolation rate of C. perfringens from aquatic animals was shown, and it was isolated from boiled clams for the first time, in which cpe and netB toxin genes were detected for the first time too. The toxin encoded by cpe gene can cause food poisoning of human, thus the discoveries of this study have certain guiding significance for food safety. Antibiotics resistant C. perfringens of aquatic origin may arise from transmission in the terrestrial environment or from antibiotic contamination of the aquaculture environment and is of public health significance.