Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

Hsa_circ_0001583 fuels bladder cancer metastasis by promoting staphylococcal nuclease and tudor domain containing 1-mediated MicroRNA decay.

Muscle-invasive and metastatic bladder cancer indicates extra worse prognosis. Accumulating evidence roots for the prominent role of circular RNAs(circRNAs) in bladder cancer, while the mechanisms linking circRNAs and bladder cancer metastasis remain limitedly investigated. Here, we identified a significantly upregulated circRNA candidate, hsa_circ_0001583, from online datasets. Validated by qRT-PCR, PCR, sanger sequencing, actinomycin D and RNase R digestion experiments, hsa_circ_0001583 was proved to be a genuine circular RNA with higher expression levels in bladder cancer tissue. Through gain and loss of function experiments, hsa_circ_0001583 exhibited potent migration and invasion powers both in vitro and in vivo. The staphylococcal nuclease and Tudor domain containing 1 (SND1) was identified as an authentic binding partner for hsa_circ_0001583 through RNA pulldown and RIP experiments. Elevated levels of hsa_circ_0001583 could bind more to SND1 and protect the latter from degradation. Rescue experiments demonstrated that such interaction-induced increased in SND1 levels in bladder cancer cells enabled the protein to pump its endonuclease activity, leading to the degradation of tumor-suppressing MicroRNAs (miRNAs) including miR-126-3p, the suppressor of Disintegrin And Metalloproteinase Domain-Containing Protein 9 (ADAM9), ultimately driving cells into a highly migrative and invasive state. In summary, our study is the first to highlight the upregulation of hsa_circ_0001583 in bladder cancer and its role in downregulating miR-126-3p by binding to and stabilizing the SND1 protein, thereby promoting bladder cancer cell migration and invasion. This study adds hsa_circ_0001583 to the pool of bladder cancer metastasis biomarkers and therapeutic targets.

Micrococcal nuclease sequencing of porcine sperm suggests enriched co-location between retained histones and genomic regions related to semen quality and early embryo development.

The mammalian spermatozoon has a unique chromatin structure in which the majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The sperm's chromatin structure remains unresolved in most animal species, including the pig. However, mapping the genomic locations of retained nucleosomes in sperm could help understanding the molecular basis of both sperm development and function as well as embryo development. This information could then be useful to identify molecular markers for sperm quality and fertility traits. Here, micrococcal nuclease digestion coupled with high throughput sequencing was performed on pig sperm to map the genomic location of mono- and sub-nucleosomal chromatin fractions in relation to a set of diverse functional elements of the genome, some of which were related to semen quality and early embryogenesis. In particular, the investigated elements were promoters, the different sections of the gene body, coding and non-coding RNAs present in the pig sperm, potential transcription factor binding sites, genomic regions associated to semen quality traits and repeat elements. The analysis yielded 25,293 and 4,239 peaks in the mono- and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison revealed positional conservation of the nucleosome retention in sperm between the pig data and a human dataset that found nucleosome enrichment in genomic regions of importance in development. Both gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and the identification of putative transcription factor binding motifs within the mono- and the sub- nucleosomal peaks showed enrichment for processes related to sperm function and embryo development. There was significant motif enrichment for Znf263, which in humans was suggested to be a key regulator of genes with paternal preferential expression during early embryogenesis. Moreover, enriched positional intersection was found in the genome between the mono-nucleosomal peaks and both the RNAs present in pig sperm and the RNAs related to sperm quality. There was no co-location between GWAS hits for semen quality in swine and the nucleosomal sites. Finally, the data evidenced depletion of mono-nucleosomes in long interspersed nuclear elements and enrichment of sub-nucleosomes in short interspersed repeat elements.These results suggest that retained nucleosomes in sperm could both mark regulatory elements or genes expressed during spermatogenesis linked to semen quality and fertility and act as transcriptional guides during early embryogenesis. The results of this study support the undertaking of ambitious research using a larger number of samples to robustly assess the positional relationship between histone retention in sperm and the reproductive ability of boars.

The Drosophila BEAF insulator protein interacts with the polybromo subunit of the PBAP chromatin remodeling complex.

The Drosophila Boundary Element-Associated Factor of 32 kDa (BEAF) binds in promoter regions of a few thousand mostly housekeeping genes. BEAF is implicated in both chromatin domain boundary activity and promoter function, although molecular mechanisms remain elusive. Here, we show that BEAF physically interacts with the polybromo subunit (Pbro) of PBAP, a SWI/SNF-class chromatin remodeling complex. BEAF also shows genetic interactions with Pbro and other PBAP subunits. We examine the effect of this interaction on gene expression and chromatin structure using precision run-on sequencing and micrococcal nuclease sequencing after RNAi-mediated knockdown in cultured S2 cells. Our results are consistent with the interaction playing a subtle role in gene activation. Fewer than 5% of BEAF-associated genes were significantly affected after BEAF knockdown. Most were downregulated, accompanied by fill-in of the promoter nucleosome-depleted region and a slight upstream shift of the +1 nucleosome. Pbro knockdown caused downregulation of several hundred genes and showed a correlation with BEAF knockdown but a better correlation with promoter-proximal GAGA factor binding. Micrococcal nuclease sequencing supports that BEAF binds near housekeeping gene promoters while Pbro is more important at regulated genes. Yet there is a similar general but slight reduction of promoter-proximal pausing by RNA polymerase II and increase in nucleosome-depleted region nucleosome occupancy after knockdown of either protein. We discuss the possibility of redundant factors keeping BEAF-associated promoters active and masking the role of interactions between BEAF and the Pbro subunit of PBAP in S2 cells. We identify Facilitates Chromatin Transcription (FACT) and Nucleosome Remodeling Factor (NURF) as candidate redundant factors.

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination.

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9's REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9's DNA- and guide RNA- interacting residues to optimize its editing fidelity.

Cooperative binding between distant transcription factors is a hallmark of active enhancers.

Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent of TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short micrococcal nuclease (MNase)-protected DNA segments indicates that the majority of enhancers harbor two or more TF-binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at the majority of active enhancers. Cooperativity is highest between sites spaced 50 bp apart, indicating that cooperativity occurs without apparent protein-protein interactions. Our findings suggest nucleosomes promoting cooperativity because co-binding may effectively clear nucleosomes and promote enhancer function.

Simultaneous electrochemical determination of nuc and mecA genes for identification of methicillin-resistant Staphylococcus aureus using N-doped porous carbon and DNA-modified MOF.

The detection of Staphylococcus aureus specific gene in combination with the mecA gene is vitally important for accurate identification of methicillin-resistant Staphylococcus aureus (MRSA). A homogeneous electrochemical DNA sensor was fabricated for simultaneous detection of mecA and nuc gene in MRSA. Metal-organic framework (type UiO-66-NH2) was applied as nanocarrier. Two electroactive dyes, methylene blue (MB) and epirubicin (EP), were encapsulated in UiO-66-NH2, respectively, and were locked by the hybrid double-stranded DNA. Based on the target-response electroactive dye release strategy, once target DNA exists, it completely hybridizes with displacement DNA (DEP and DMB). So DEP and DMB is displaced from the MOF surface, causing the release of electroactive dyes. Co-Zn bimetallic zeolitic imidazolate framework-derived N-doped porous carbon serves for electrode modification to improve electrocatalytic performance and sensitivity. The differential pulse voltammetry peak currents of MB and EP were accurately detected at - 0.14 V and - 0.53 V versus the Ag/AgCl reference electrode, respectively. Under the optimal conditions, the detection limits of mecA gene and nuc gene were 3.7 fM and 1.6 fM, respectively. Combining the effective application of MOFs and the homogeneous detection strategy, the sensor exhibited satisfactory performance for MRSA identification in real samples. The recovery was 92.6-103%, and the relative standard deviation was less than 5%. Besides, MRSA and SA can also be distinguished. This sensor has great potential in practical applications.

Development and application of a triplex-PCR assay for rapid detection of methicillin-resistant Staphylococcus aureus from pigs.

A triplex-PCR assay was developed and evaluated for rapid detection of methicillin-resistant Staphylococcus aureus (MRSA) recovered from various biological samples of pig. Three sets of primers were designed to target mecA, 16S rRNA and nuc genes of MRSA. The specific amplification generated three bands on agarose gel, with sizes 280 bp for mecA, 654 bp for 16S rRNA and 481 bp for nuc, respectively. A potential advantage of the PCR assay is its sensitivity with a detection limit of 102  CFU per ml of bacteria. In all, 79 MRSA isolates recovered from various samples of pigs were subjected to the amplification by the triplex-PCR assay and all the isolates yielded three bands corresponding to the three genes under this study. No false-positive amplification was observed, indicating the high specificity of the developed triplex-PCR assay. This assay will be a useful and powerful method for differentiation of MRSA from methicillin-sensitive S. aureus, coagulase-negative methicillin-resistant staphylococci and coagulase-negative methicillin-sensitive staphylococci.

Micrococcal Nuclease stimulates Staphylococcus aureus Biofilm Formation in a Murine Implant Infection Model.

Advancements in contemporary medicine have led to an increasing life expectancy which has broadened the application of biomaterial implants. As each implant procedure has an innate risk of infection, the number of biomaterial-associated infections keeps rising. Staphylococcus aureus causes 34% of such infections and is known as a potent biofilm producer. By secreting micrococcal nuclease S. aureus is able to escape neutrophil extracellular traps by cleaving their DNA-backbone. Also, micrococcal nuclease potentially limits biofilm growth and adhesion by cleaving extracellular DNA, an important constituent of biofilms. This study aimed to evaluate the impact of micrococcal nuclease on infection persistence and biofilm formation in a murine biomaterial-associated infection-model with polyvinylidene-fluoride mesh implants inoculated with bioluminescent S. aureus or its isogenic micrococcal nuclease deficient mutant. Supported by results based on in-vivo bioluminescence imaging, ex-vivo colony forming unit counts, and histological analysis it was found that production of micrococcal nuclease enables S. aureus bacteria to evade the immune response around an implant resulting in a persistent infection. As a novel finding, histological analysis provided clear indications that the production of micrococcal nuclease stimulates S. aureus to form biofilms, the presence of which extended neutrophil extracellular trap formation up to 13 days after mesh implantation. Since micrococcal nuclease production appeared vital for the persistence of S. aureus biomaterial-associated infection, targeting its production could be a novel strategy in preventing biomaterial-associated infection.

pH-Dependent Conformational Changes Lead to a Highly Shifted pKa for a Buried Glutamic Acid Mutant of SNase.

Ionizable residues are rarely present in the hydrophobic interior of proteins, but when they are, they play important roles in biological processes such as energy transduction and enzyme catalysis. Internal ionizable residues have anomalous experimental pKa values with respect to their pKa in bulk water. This work investigates the atomistic cause of the highly shifted pKa of the internal Glu23 in the artificially mutated variant V23E of Staphylococcal Nuclease (SNase) using pH replica exchange molecular dynamics (pH-REMD) simulations. The pKa of Glu23 obtained from our calculations is 6.55, which is elevated with respect to the glutamate pKa of 4.40 in bulk water. The calculated value is close to the experimental pKa of 7.10. Our simulations show that the highly shifted pKa of Glu23 is the product of a pH-dependent conformational change, which has been observed experimentally and also seen in our simulations. We carry out an analysis of this pH-dependent conformational change in response to the protonation state change of Glu23. Using a four-state thermodynamic model, we estimate the two conformation-specific pKa values of Glu23 and describe the coupling between the conformational and ionization equilibria.

Genotypic and phenotypic characterisation of clinical isolates of methicillin-resistant Staphylococcus aureus in two different geographical locations of Iran.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) has become more prevalent all over the world and it is important to determine MRSA prevalence and typing in different regions. The present study was carried out to determine the prevalence and frequency of circulating molecular types of MRSA isolates as well as their antibiotics susceptibility in Tabriz and Kerman cities of Iran. Materials and Methods: A total of 230 S. aureus isolates were collected from Tabriz (n=125) and Kerman (n=105) during January to December 2018. MRSA isolates were identified by PCR amplification of nuc and mec A genes. Antibiotic susceptibility of MRSA isolates were determined by Kirby-Bauer disk diffusion method. Multiplex PCR was exploited to detect various types of SCCmec. Results: The MRSA prevalence was 51/125 (40.8%) in Tabriz and 60/105 (57.1%) in Kerman. Overall, 36/51 (70.58%) and 15/51 (29.41%) isolates and 37/60 (61.66%) and 23/60 (38.34%) isolates were isolated from inpatients and outpatients in Tabriz and Kerman, respectively. Almost all of the isolates were resistant to penicillin and all of them were sensitive to linezolid. Thirty five (68.2%) and 34(56.6%) of MRSA isolates in Tabriz and Kerman were determined as MDR, respectively. SCCmec typing showed that the frequent SCCmec type in both Tabriz and Kerman cities was SCCmec III (56.86% and 55%, respectively). Conclusion: The high prevalence of MRSA makes it necessary to revisit the antibiotics administration by physicians. Indeed, periodic evaluation of antibacterial susceptibility patterns of the MRSA strains is required for efficient treatment of MRSA infections.

Nasal carriage of Staphylococcus aureus in farm animals and breeders in north of Morocco.

BACKGROUND: The objectives of this study were to determine for the first time, in Morocco, the nasal carriage rate, antimicrobial susceptibility profiles and virulence genes of Staphylococcus. aureus isolated from animals and breeders in close contact. METHODS: From 2015 to 2016, 421 nasal swab samples were collected from 26 different livestock areas in Tangier. Antimicrobial susceptibility phenotypes were determined by disk diffusion according to EUCAST 2015. The presence of nuc, mecA, mecC, lukS/F-PV, and tst genes were determined by Polymerase Chain Reaction (PCR) for all isolates. RESULTS: The overall S. aureus nasal carriage rate was low in animals (9.97%) and high in breeders (60%) with a statistically significant difference, (OR = 13.536; 95% CI = 7.070-25.912; p < 0.001). In general, S. aureus strains were susceptible to the majority of antibiotics and the highest resistance rates were found against tetracycline (16.7% in animals and 10% in breeders). No Methicillin-Resistant S. aureus (MRSA) was detected in animals and breeders. A high rate of tst and lukS/F-PV genes has been recovered only from animals (11.9 and 16.7%, respectively). CONCLUSION: Despite the lower rate of nasal carriage of S. aureus and the absence of MRSA strains in our study, S. aureus strains harbored a higher frequency of tst and lukS/F-PV virulence genes, which is associated to an increased risk of infection dissemination in humans. This highlights the need for further larger and multi-center studies to better define the transmission of the pathogenic S. aureus between livestock, environment, and humans.

Leukocidins and the Nuclease Nuc Prevent Neutrophil-Mediated Killing of Staphylococcus aureus Biofilms.

Bacterial biofilms are linked with chronic infections and have properties distinct from those of planktonic, single-celled bacteria. The virulence mechanisms associated with Staphylococcus aureus biofilms are becoming better understood. Human neutrophils are critical for the innate immune response to S. aureus infection. Here, we describe two virulence strategies that converge to promote the ability of S. aureus biofilms to evade killing by neutrophils. Specifically, we show that while neutrophils exposed to S. aureus biofilms produce extracellular traps (NETs) and phagocytose bacteria, both mechanisms are inefficient in clearance of the biofilm biomass. This is attributed to the leukocidin LukAB, which promotes S. aureus survival during phagocytosis. We also show that the persistence of biofilm bacteria trapped in NETs is facilitated by S. aureus nuclease (Nuc)-mediated degradation of NET DNA. This study describes key aspects of the interaction between primary human neutrophils and S. aureus biofilms and provides insight into how S. aureus evades the neutrophil response to cause persistent infections.

Short communication: Virulence profiles of Staphylococcus aureus isolated from bulk tank milk and adherences on milking equipment on Chilean dairy farms.

Staphylococcus aureus is an important intramammary pathogen for dairy cows that also is remarkably important for public health. Multiple virulence factors can be involved simultaneously during the pathogenesis of a staphylococcal disease, including adhesion proteins, extracellular enzymes, and toxins. The main objective of this study was to assess virulence factors that are associated with cow intramammary infection (IMI) and of human health concern among Staph. aureus isolates obtained from bulk tank milk (BTM) and adherences on milking equipment surfaces. A total of 166 Staph. aureus isolates from 23 dairy farms were characterized according to their virulence profiles. For virulence factors of importance in IMI, the presence of the virulence markers thermonuclease (nuc) and coagulase (coa) and virulence genes such as fibronectin (fnbA) and intercellular adhesion (icaA, icaD) were assessed. For virulence factors of public health concern, presence of antimicrobial resistance (mecA and mecC) and enterotoxin (sea and seb) genes were analyzed. Among all Staph. aureus isolates, 5 virulence profiles were found; the profile nuc(+)coa(+)fnbA(+)icaA(+)icaD(+)mecA(-)mecC(-)sea(-)seb(-) was the most frequently observed (21 out of 23 dairy farms). No differences were found between the virulence profile frequencies of Staph. aureus from BTM and adherences on milking equipment surfaces. The virulence profiles most frequently observed included genes involved in the adherence and biofilm-forming ability of Staph. aureus, which could represent a potential advantage for the bacterium during the early stages of IMI colonization and for persistence on surfaces. Our results indicate a greater frequency of virulence factors of importance for IMI pathogenesis than virulence factors of public health concern, consistent with the dairy origin of isolates. The mecA, mecC, and seb genes were not observed among Staph. aureus isolates analyzed in this study. However, the sea gene was detected in 3 Staph. aureus isolated from BTM, thus posing a potential public health threat. Our results emphasize the importance of understanding the epidemiology and dynamics of Staph. aureus on dairy farms as a tool for the improvement of udder health and milk safety.

Thermonuclease test accuracy is preserved in methicillin-resistant Staphylococcus aureus isolates.

Introduction. The nuc gene encodes a thermonuclease which is present in Staphylococcus aureus but not in coagulase-negative staphylococci (CoNS) and is the target of the rapid phenotypic thermonuclease test. The effect of nuc gene variation in methicillin-resistant S. aureus (MRSA) on the performance of PCR testing has been noted, although there are no reports about the effect of MRSA on the activity of the thermonuclease enzyme.Aim. Our goals were to examine the sensitivity and specificity of the thermonuclease test used to distinguish S. aureus from CoNS cultured from blood. In addition, we aimed to assess differences in the sensitivity, specificity and accuracy of the thermonuclease test between methicillin-sensitive S. aureus (MSSA) and MRSA isolates.Methodology. We performed a retrospective analysis of 1404 isolates. Each isolate from a positive blood culture was identified as a Gram-positive coccus by microscopy then analysed with the thermonuclease test (Southern Group Laboratory) prior to confirmatory identification using VITEK microbial identification platforms (bioMérieux) and cefoxitin disc diffusion testing.Results. Of 1331 samples included in the final analysis, 189 were thermonuclease-positive, of which 176 were identified as S. aureus. Of the 1142 thermonuclease-negative samples, 13 were finally identified as S. aureus, giving a sensitivity of 93.1 % (95 % confidence interval [CI] 88.5-96.3) and specificity of 98.9 % (95 % CI 98.1-99.4). Of the nine proven MRSA samples, eight were thermonuclease-positive, giving a sensitivity of 88.9 % (95 % CI 51.8-99.7). Thermonuclease test accuracy for MSSA and MRSA isolates was 98.1 % (95 % CI 97.2-98.8) versus 98.8 % (95 % CI 98.0-99.3), respectively.Conclusions. In the era of increasing use of molecular-based microbiology assays, the thermonuclease test remains a simple, inexpensive and robust test for the presumptive identification of S. aureus cultured from blood, irrespective of methicillin sensitivity.

Antibacterial photodynamic therapy mediated by 5-aminolevulinic acid on methicillin-resistant Staphylococcus aureus.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), has brought great difficulties to clinical treatment. Antibacterial photodynamic therapy (aPDT) is a new non-antibiotic treatment strategy for a variety of drug-resistant bacteria. However, there are few studies on the antimicrobial mechanism of a PDT mediated by 5-aminolevulinic acid (ALA-PDT) for MRSA. In this study, we observed the bactericidal effect of ALA-PDT on MRSA. We found that ALA-PDT had the strongest bactericidal effect when ALA was at 0.05 mM, and the bactericidal activity of aPDT increased with the increase of light dose. MRSA was almost completely eliminated at 0.05 mM and 384 Jcm-2. In addition, the bactericidal morphology was also observed under a fluorescence microscope using a LIVE/DEAD® BacLight™ Bacterial Viability Kit and an electron microscope. It was also found that proteins and DNA were damaged by ALA-PDT. Finally, the transcription level of the specific gene of MRSA, nuc, was decreased by 0.74-fold (P < 0.05) after ALA-PDT treatment by qRT-PCR analysis. The findings suggest that ALA-PDT can effectively inhibit MRSA by damaging cell membrane, cytoplasm, proteins and nucleic acid.

Therapeutic effects of duck Tembusu virus capsid protein fused with staphylococcal nuclease protein to target Tembusu infection in vitro.

Tembusu virus (TMUV), a member of the genus flavivirus, primarily causes egg-drop syndrome in ducks and is associated with low disease mortality but high morbidity. The commercially available live vaccines for treating TMUV currently include the main WF100, HB, and FX2010-180P strains, and efficient treatment and/or preventative measures are still urgently needed. Capsid-targeted viral inactivation (CTVI) is a conceptually powerful new antiviral strategy that is based on two proteins from the capsid protein of a virus and a crucial effector molecule. The effector molecule can destroy the viral DNA/RNA or interfere with the proper folding of key viral proteins, while the capsid protein mainly plays a role in viral integration and assembly; the fusion proteins are incorporated into virions during packaging. This study aimed to explore the potential use of this strategy in duck TMUV. Our results revealed that these fusion proteins can be expressed in susceptible BHK21 cells without cytotoxicity and possess excellent Ca2+-dependent nuclease activity, and their expression is also detectable in DF-1 cells. Compared to those in the negative controls (BHK21 and BHK21/pcDNA3.1(+) cells), the numbers of viral RNA copies in TMUV-infected BHK21/Cap-SNase and BHK21/Cap-Linker-SNase cells were reduced by 48 h, and the effect of Cap-Linker-SNase was superior to that of Cap-SNase. As anticipated, these results suggest that these fusion proteins contribute to viral resistance to treatment. Thus, CTVI might be applicable for TMUV inhibition as a novel antiviral therapeutic candidate during viral infection.

Vancomycin-resistant Staphylococcus aureus isolated from camel meat and slaughterhouse workers in Egypt.

Background: The emergence of vancomycin-resistant Staphylococcus aureus (VRSA) represents a challenge for the treatment of staphylococcal infections in both human and animals worldwide. Although VRSA has been detected in several animal species worldwide, data on the bacterial prevalence in dromedary camels and workers in camel slaughterhouses are scarce. Methods: We investigated meat samples from 200 dromedary camel carcasses from three different abattoirs that were being prepared to be sent to the markets. Twenty hand swabs were voluntarily collected from the workers in the same abattoirs. Isolation and identification of the bacterial specimens from the samples were performed using conventional cultural techniques and biochemical identification and were confirmed by PCR amplification of the nuc gene. Antimicrobial susceptibility against nine antimicrobial agents commonly used in human and camels was tested using the disc diffusion method, and genetic analysis was performed by evaluating the mecA gene in phenotypically oxacillin (OXA)- and cefoxitin (FOX)-resistant isolates. The resistance of S. aureus to vancomycin (VAN) was tested by broth microdilution and confirmed by PCR targeting the vanA and vanB genes. The vanA and vanB genes were sequenced. Result: S. aureus was detected in both camel meat (29/200, 14.5%) and in abattoir workers (11/20, 55%). Of the collected samples, 27% (8/29, camel) and 54% (6/11, human) were identified as VRSA.All VRSA isolates carried both the vanA and vanB genes. Additionally, all VRSA isolates were also classified as methicillin-resistant S. aureus (MRSA). The vanA amplicons of the isolates from human and camel meat were homologous and clustered with a Chinese reference isolate sequence. Conclusion: This study demonstrated that VRSA is present in camel abattoirs in Egypt. Zoonotic transmission between animals and human is probable and reflects both a public health threat and a food safety concern.

Mycoplasma hyopneumoniae Mhp597 is a cytotoxicity, inflammation and immunosuppression associated nuclease.

Nucleases are ubiquitously recognized as essential proteins in mycoplasmas because these organisms lack most capacities for de novo synthesis of nucleotides. Some of these proteins were proved to be important pathogenic factors during the infection of mycoplasms. In this study, the protein Mhp597 from M. hyopneumoniae was expressed and purified in Escherichia coli. Analysis of nuclease activity showed that recombinant Mhp597 (rMhp597) was a Ca2+ or Mg2+ dependent thermostable nuclease with very high activity and neutrophil extracellular traps (NETs) induced by M. hyopneumoniae were completely degraded by this nuclease. In addition, when PK15 cells were incubated with different concentrations of rMhp597, their viability was reduced and cell apoptosis was observed in a dose-dependent manner. To further investigate the host immune system response, we report that rMhp597 up-regulated the exression of inflammatory genes showing that TLR4/MyD88/NF-κB signal pathway was involved. On the other hand, rMhp597 down-regulated the expression of type I IFN (IFN-α/ß) and promoted the multiplication of porcine reproductive and respiratory syndrome virus (PRRSV). Recombinant rMhp597δ315-377 lacking C-terminal 63 amino acids exhibited all biological functions mentioned above except for nuclease activity. In summary, Mhp597 is a dynamic secreted nuclease involved in cytotoxicity, inflammation and immunosuppression.

A 13-Plex PCR for high-resolution melting-based screening of clinically important Staphylococcus species in animals.

A single-tube multiplex real-time PCR targeting the nuclease (nuc) gene and subsequent high-resolution melting analysis (HRMA) were used to identify 13 different Staphylococcus species. The nuc gene was targeted due to its low intraspecies variation and the greater interspecies variation than the 16S rRNA gene in Staphylococcus. We used HRMA software that can store and compare HRMA profiles from different runs as long as the runs contain the same reference reaction. Thus, we reduced the 14 PCRs to 2 different PCRs, one targeting the unknown sequence and the other targeting the reference sequences to screen 13 different Staphylococcus species. The specificity of the developed method was tested on 16 different Staphylococcus reference strains and 115 different field strains that were isolated from the milk of cattle with subclinical mastitis. We conclude that the method can be used to quickly and cost-effectively differentiate Staphylococcus aureus (S. aureus) from other Staphylococcus species (S. epidermidis, S. lugdunensis, S. schleiferi, S. hyicus, S. chromogenes, S. lentus, S. haemolyticus, S. xylosus, S. saprophyticus, S. warneri, S. simulans and S. hominis).