The Relative Importance of Cytotoxins Produced by Methicillin-Resistant Staphylococcus aureus Strain USA300 for Causing Human PMN Destruction.

Staphylococcus aureus (S. aureus) is a prominent Gram-positive bacterial pathogen that expresses numerous cytotoxins known to target human polymorphonuclear leukocytes (PMNs or neutrophils). These include leukocidin G/H (LukGH, also known as LukAB), the Panton-Valentine leukocidin (PVL), γ-hemolysin A/B (HlgAB), γ-hemolysin B/C (HlgBC), leukocidin E/D (LukED), α-hemolysin (Hla), and the phenol-soluble modulin-α peptides (PSMα). However, the relative contribution of each of these cytotoxins in causing human PMN lysis is not clear. In this study, we used a library of cytotoxin deletion mutants in the clinically relevant methicillin-resistant S. aureus (MRSA) isolate LAC (strain ST8:USA300) to determine the relative importance of each for causing human PMN lysis upon exposure to extracellular components as well as following phagocytosis. Using flow cytometry to examine plasma membrane permeability and assays quantifying lactose dehydrogenase release, we found that PVL was the dominant extracellular factor causing human PMN lysis produced by USA300. In contrast, LukGH was the most important cytotoxin causing human PMN lysis immediately following phagocytosis with contributions from the other bicomponent leukocidins only observed at later time points. These results not only clarify the relative importance of different USA300 cytotoxins for causing human PMN destruction but also demonstrate how two apparently redundant virulence factors play distinctive roles in promoting S. aureus pathogenesis.

Impact of Accidental High or Low Implantation Depth on Peri-Procedural Outcomes after Implantation with the Self-Expanding ACURATE neo2.

Background: Precise implantation could play a crucial role in the technical success of transcatheter aortic valve replacement (TAVR) for some prostheses. The impact of an accidental implantation depth (ID) outside the recommended range has not been assessed for the ACURATE neo2 (NEO2). Methods: Data from 1839 patients with severe native aortic stenosis treated with the NEO2 prosthesis were evaluated. We compared the results of prostheses implanted in an ID both inside and outside the recommendations. The outcome assessment followed the Valve Academic Research Consortium-3 criteria. Results: Patients were retrospectively divided into high (<3 mm; n = 412), optimal (3-7 mm; n = 1236), and low (>7 mm; n = 169) implantations. Technical success (94.7% vs. 94.7% vs. 91.7%, p = 0.296) and device success were high (90.1% vs. 89.3% vs. 84.6%, p = 0.112) without differences between groups. Rates of relevant paravalvular regurgitation (PVL; >mild or VinV due to PVL) were comparable (1.2% vs. 1.8% vs. 1.2%, p = 0.759). Even when hemodynamics were superior in the high-implantation group, with greater iEOA (1.01 cm2/m2 vs. 0.95 cm2/m2 vs. 0.92 cm2/m2, p < 0.001), spontaneous embolization or after post-dilatation was more common. Low implantation was associated with a higher rate of associated pacemaker implantation (PPI) (6.1% vs. 8.8% vs. 14.8%, p = 0.001). Conclusions: Implantation with the ACURATE neo2 showed excellent hemodynamic results, including low gradients and a small number of relevant PVL, in line with a high technical success rate that was irrespective of the ID. A favorable outcome can also be achieved in accidental low or high positions. Low implantation was associated with a higher rate of associated pacemaker implantation. Deliberately high implantation should be avoided due to the risk of embolization.

Necrotizing pneumonia secondary to Influenza A (H1N1) coinfection with Staphylococcus aureus: A case report.

Objective: This study aims to enhance understanding of necrotizing pneumonia and toxic shock syndrome by analyzing an adult case of community-acquired necrotizing pneumonia caused by co-infection of Influenza A (H1N1) and Staphylococcus aureus with LukS-PV and LukF-PV virulence factor genes. Method: The clinical data of one patient admitted to the intensive care unit (ICU) with co-infection of Influenza A (H1N1) and Staphylococcus aureus was retrospectively analyzed. Results: The patient exhibited typical clinical manifestations of viral and Staphylococcus aureus co-infection, including necrotizing pneumonia and toxic shock syndrome. The presence of LukS-PV and LukF-PV virulence factor genes of Staphylococcus aureus was detected in the patient's bronchoalveolar lavage fluid. Unfortunately，although antiviral agents (oseltamivir) and antibiotics (linezolid, imipenem-cilastatin) were timely administrated, as well as corticosteroids for anti-inflammatory purposes, the patient's condition was progressively deteriorated and eventually led to death. Conclusion: Clinical practitioners should be vigilant about the co-infection of Influenza virus and Staphylococcus aureus, particularly when the latter carries virulence factors. The presence of virulence factor genes of Staphylococcus aureus can lead to necrotizing pneumonia with a poor prognosis. This is a particular concern because both infections can be life threatening in young adults.

Panton-Valentine leucocidin gene in methicillin resistant Staphylococcus aureus isolated from tertiary care hospital in Nepal.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) expresses the Panton-Valentine leukocidin (PVL) virulence gene, which is associated with community and hospital-acquired severe MRSA infections. The objective of this study was to determine the prevalence and antibiotic susceptibility profile with a focus on the presence of the PVL gene among MRSA isolates in healthcare settings. METHODOLOGY: A total of 1,207 clinical specimens and 304 hospital environment swabs were collected in a tertiary care hospital in Nepal, and investigated following basic microbiological techniques. S. aureus was confirmed with the coagulase test. An antibiotic susceptibility test (AST) was performed by the Kirby-Bauer method and screening for MRSA was carried out by the cefoxitin disc diffusion method guided by the Clinical and Laboratory Standards Institute (CLSI), 2020. DNA was extracted and used in a polymerase chain reaction (PCR) to detect mecA and PVL genes. RESULTS: Of the 1,511 samples, 45 (2.9%) S. aureus (23 clinical and 22 environmental) were isolated. Among them, 69.6% (16/23) and 27.3% (6/22) were MRSA in clinical and environmental isolates, respectively. Twelve (52.2%) clinical isolates and seven (31.8%) environmental isolates were multidrug resistant. The majority of isolates were susceptible to vancomycin and linezolid. The PVL gene was detected in 18.2% (n = 4/22) of the MRSA isolates, of which three were from clinical sources and one was from an environmental swab. CONCLUSIONS: The prevalence of MRSA, and PVL-producing S. aureus were higher in the hospital setting. Hence, immediate and urgent implementation of infection control and sanitation measures are needed in the hospital.

A Single Lung Abscess Caused by Panton-Valentine Leukocidin-Producing Methicillin-Resistant Staphylococcus aureus.

This case report presents a rare occurrence of a single lung abscess caused by Panton-Valentine leukocidin (PVL)-producing methicillin-resistant Staphylococcus aureus (MRSA) in a 38-year-old immunocompetent man. The patient, of Southeast Asian origin, presented with symptoms of fever, chest pain, cough, and shortness of breath following a recent flu-like illness. Imaging indicated a cavitary lung lesion in the left lower lobe, suggestive of a lung abscess. Initial antibiotic treatment failed, and drainage of the abscess confirmed MRSA with the PVL gene, indicating a community-acquired MRSA infection. The patient received intravenous vancomycin followed by oral linezolid, leading to the resolution of the abscess. Contact tracing and decolonization measures were implemented. This case highlights the importance of considering PVL-producing S. aureus as a potential pathogen in severe necrotizing pneumonia or sepsis and underscores the need for prompt diagnosis, appropriate antibiotic therapy, and infection control measures in managing such infections.

Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Family and Pet Cat.

Continued detection of Panton-Valentine leukocidin-positive Staphylococcus aureus in samples from a family with severe repeated skin infections and their pet cat suggests transmission between the family and the cat. Decolonizing the pet led to successful elimination of the bacteria from the household. Clinicians should consider pet cats as possible reinfection sources.

Investigation of SCCmec types using the real time PCR method in cefoxitin-resistant Staphylococcus aureus isolates.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen that can cause many community and hospital-acquired infections. This study was conducted to investigate the SCCmec gene types responsible for methicillin resistance in MRSA isolates isolated from hospitalised patients. MATERIAL AND METHODS: MRSA isolates isolated from samples sent from various clinics to Gaziantep University Hospital Microbiology Laboratory between March 2021-January 2022 were included in the study. Bacteria were identified using by VITEK 2 automated system. Cefoxitin (FOX) resistance was determined by the disc diffusion method according to EUCAST standards. Cefoxitin resistance was confirmed by the Penicillin Binding Protein 2' latex agglutination test. Types of mecA, mecC, coa, nuc, Panton Valentin Leukocidin (PVL), ccrC2, class A mec, SCCmec types in isolates detected as MRSA were investigated by real-time PCR. RESULTS: In this study, 116 isolates meeting the study criteria were examined. By detecting the nuc and coa genes in all isolates by PCR, the phenotypic identification of S.aureus was confirmed. While the mecA gene was detected in all MRSA isolates, no mecC gene was detected in any isolates. Detected SCCmec types were as follows; SCCmec Type 1 (2.6%), Type II (28.4%), Type III (12.9%), Type IVa (11.2%), Type IVb (3.4%), Type IVc (3.4%), Type IVg (12.1%), Type V (0.9%), Type VII (4.3%), Type VIII (18.1%), Type IX (0.9%), Type XII (1.7%). On the other hand, SCCmec Type VI, X, XI and XIII were not found in any isolate. It was determined that four of the MRSA isolates (3.4%) carried the PVL gene that two (50%) of these were found in SCCmec Type VIII. CONCLUSION: Monitoring of FOX resistance is an effective and safe method for determination of MRSA isolates. The change in the mec gene causes resistance, which should be monitored regularly with molecular methods. Our study is the first study in Turkey.

Do we still need intra-procedural TTE during Transcatheter aortic valve replacement? A high volume, single center experience.

BACKGROUND: Transcatheter aortic valve implantation (TAVI) has undergone significant advances in recent years, with the development of improved pre-planning tools and devices. These advances have led to a reduction in the rate of paravalvular leak (PVL), a complication that is associated with poor outcomes even when mild. As some centers around the world are moving to solely fluoroscopy-focused implantation, we aimed to describe the clinical impact of intra-procedural transthoracic echocardiography (TTE) during TAVI in a high volume hospital. METHODS: Observational study during a 3-month period. A limited TTE examination was performed immediately after deployment to assess the existence of PVL and grade its severity. Complete TTE was performed a day after the procedure. In case of ≥mild PVL after valve deployment, a decision was made according to the severity of the PVL, patient anatomy and extent of annular calcification to preform balloon post-dilation. If done, an additional limited TTE was performed to assess possible complication and the degree of PVL post dilatation. RESULTS: 115 patient were included in the study. Intra-procedural TTE identified 16 patients (14 %) with at least mild PVL, three of them with moderate (3 %). Post balloon dilatation was performed in 10 patients (9 % of the cohort) with significant improvement in the degree of PVL. CONCLUSION: Intra-procedural TTE immediately after TAVI deployment can accurately identify PVL, allowing operators to perform post balloon dilatation with improvement in early echocardiographic results. Our findings support the routine use of TTE during procedures, without relying solely on fluoroscopy.

Genotypic insights to Panton-Valentine leukocidin positive methicillin-resistant Staphylococcus aureus isolated from cattle mastitis.

In this research communication we investigate the prevalence and antimicrobial susceptibility of S. aureus harboring virulent genes responsible for mastitis in cattle of Punjab, Pakistan. A total of 690 milk samples were collected from commercial dairy farms for analysis of the prevalence of subclinical and clinical mastitis and isolation of S. aureus. Virulence ability and methicillin resistance in S. aureus (MRSA) was determined by targeting the pvl (the gene for Panton-Valentine leukocidin) and mecA genes, respectively. A total of 175 S. aureus isolates exhibiting prevalence of pvl gene (6.28%) and mecA gene (22.28%) were determined. Antimicrobial susceptibility testing of pvl positive and negative MRSA against different classes of antibiotics revealed 100% resistance against ß-lactams while 100% sensitivity towards tylosin and linezolid.

Like Father, Like Daughter - Ectodermal Dysplasia-Syndactyly Syndrome: A Case Report.

Ectodermal dysplasia-syndactyly syndrome 1 (EDSS1) is an exceedingly rare condition associated with mutations in the PVL4 gene. It is characterised by sparse, brittle hair, eyebrows and eyelashes, abnormal dentition and nails, along with bilateral cutaneous syndactyly involving the fingers and toes. We report a 2-year-old girl who presented to us with bilateral complete simple syndactyly of the third and fourth web spaces of the hands, along with bilateral syndactyly of both feet involving the second to fourth toes. Upon examination, sparse hair and eyebrows, along with abnormal dentition, were noted. Thorough clinical examination and genetic analysis were conducted on the affected child and her father, who exhibited similar clinical features. Genetic analysis revealed a homozygous nonsense mutation in the PVL4 gene in both individuals. According to the literature, EDSS1 has been reported in only 10 families worldwide, and there are no reported cases from India. Level of Evidence: Level V (Therapeutic).

A case of community-acquired Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (MRSA) necrotizing pneumonia successfully treated with two anti-MRSA drugs.

A 22-year-old Vietnamese man was referred to our hospital owing to cough, dyspnea, and difficulty moving. The patient was diagnosed with community-acquired Panton-Valentine leukocidin-positive methicillin-resistant Staphylococcus aureus (MRSA) bacteremia and necrotizing pneumonia. Treatment involved vancomycin (VCM) and meropenem, and the MRSA bacteremia improved. However, lung tissue destruction progressed. Therefore, linezolid was added to the VCM regimen, and this intervention led to the patient's recovery, and he was discharged from the hospital. Here, we report a case in which the patient was treated with a combination of two anti-MRSA drugs and was cured.

Experience with the ACURATE neo and neo2 transcatheter aortic valves in Spain. The PRECISA (PRospective Evaluation Complementing Investigation with ACURATE devices) registry.

BACKGROUND: Previous studies have documented a high rate of implantation success with the ACURATE neo2 valve, as well as a reduction in paravalvular leak (PVL) compared to its predecessor, the ACURATE neo. However, there are no studies that have reviewed and compared the long-term clinical and hemodynamic outcomes of these patients. AIMS: This study aimed to evaluate the results of the ACURATE neo transcatheter aortic valve in a real-world context, and to compare the results of the outcomes of both generations of this device (ACURATE neo and ACURATE neo2), with a specific focus on procedural success, safety, and long-term effectiveness. METHODS: A prospective study including all consecutive patients treated with the ACURATE neo device in seven hospitals was conducted (Clinical Trials Identification Number: NCT03846557). The primary endpoint consisted of a composite of adverse events, including mortality, aortic insufficiency, and other procedural complications. As the second-generation device (ACURATE neo2) replaced the ACURATE neo during the study period, hemodynamic and clinical results before admission, at 30 days, and at 1 year of follow-up were compared between the two generations. RESULTS: A total of 296 patients underwent transcatheter aortic valve implantation with the ACURATE device, with 178 patients receiving the ACURATE neo and 118 patients receiving the ACURATE neo2. In the overall population, the absence of device success occurred in 14.5%. The primary reason for the absence of device success was the presence of para-valvular regurgitation ≥ 2. There were no instances of coronary occlusions, valve embolization, annulus rupture, or procedural deaths. ACURATE neo2 was associated with a significantly higher device success rate (91.7% vs. 82%, p = 0.04), primarily due to a significantly lower rate of para-valvular regurgitation, which remained significant at 1 year. CONCLUSION: The use of ACURATE neo and neo2 transcatheter aortic valves is associated with satisfactory clinical results and an extremely low rate of complications. The ACURATE neo2 enables a significantly higher device success rate, primarily attributed to a significant reduction in the rate of PVL.

Clinical impact and public health challenges of a PVL-MRSA bacteraemia outbreak amongst people who inject drugs in South Yorkshire, UK.

Background: Panton-Valentine leukocidin (PVL) Staphylococcus aureus (SA) is an emergent public health concern. PVL toxin has been mostly associated with methicillin-sensitive S. aureus (MSSA)-related skin and soft tissue infections occurring in high-risk groups such as people who inject drugs (PWID). The emergence of PVL methicillin-resistant S. aureus (MRSA) infection is causing severe and life-threatening disease in PWID. Clinical cases: We present an outbreak of eight PVL-MRSA bacteraemia cases at a UK teaching hospital between 2018 and 2022. An additional four patients developed bacteraemia with PVL-negative MRSA of the same multilocus sequence type (MLST). All patients were PWID and aged 33-51 years old. Four patients developed MRSA bacterial endocarditis. Three patients died. These cases represent the initial cases detected at Doncaster and Bassetlaw Teaching Hospitals of what is an ongoing and developing outbreak. Management: An outbreak investigation has been undertaken in association with the UK Health Security Agency. Epidemiological factors have been explored, including via direct contact at a local sheltered accommodation and the possibility of a contaminated drug supply. Whole-genome sequencing confirmed that all isolates were closely related and of the same MLST (sequence type 5). A community substance misuse group disseminated health education on the prevention of PVL-MRSA. Preventing infection in PWID presents a major challenge due to the impact of addiction on engagement with services and the significant barriers faced by our patients in observing infection prevention measures. Conclusion: PVL-MRSA is of major public health concern and outbreak investigation and mapping out local epidemiological patterns plays a vital role in preventing further spread throughout the community. Additionally, this work enables targeted and early treatment in patients in high-risk categories for disease. These cases of PVL-MRSA infection in PWID highlights the transmissibility, pathogenic potential and severe clinical disease spectrum within this population. Further work is required to tackle transmission and infection from this pathogenic strain.

Detection of Staphylococcus aureus virulence gene pvl based on CRISPR strip.

Introduction: Staphylococcus aureus (S. aureus) is a prominent pathogen responsible for both hospital-acquired and community-acquired infections. Among its arsenal of virulence factors, Panton-Valentine Leucocidin (PVL) is closely associated with severe diseases such as profound skin infections and necrotizing pneumonia. Patients infected with pvl-positive S. aureus often exhibit more severe symptoms and carry a substantially higher mortality risk. Therefore, it is crucial to promptly and accurately detect pvl-positive S. aureus before initiating protective measures and providing effective antibacterial treatment. Methods: In this study, we propose a precise identification and highly sensitive detection method for pvl-positive S. aureus based on recombinase-assisted amplification and the CRISPR-ERASE strip which we previously developed. Results: The results revealed that this method achieved a detection limit of 1 copy/µL for pvl-positive plasmids within 1 hour. The method successfully identified all 25 pvl-positive and 51 pvl-negative strains among the tested 76 isolated S. aureus samples, demonstrating its concordance with qPCR. Discussion: These results show that the CRISPR-ERASE detection method for pvl-positive S. aureus has the advantages of high sensitivity and specificity, this method combines the characteristics of recombinase-assisted amplification at room temperature and the advantages of ERASE test strip visualization, which can greatly reduce the dependence on professional laboratories. It is more suitable for on-site detection than PCR and qPCR, thereby providing important value for rapid on-site detection of pvl.

Staphylococcus aureus Panton-Valentine Leukocidin worsens acute implant-associated osteomyelitis in humanized BRGSF mice.

Staphylococcus aureus is the most common pathogen that causes implant-associated osteomyelitis, a clinically incurable disease. Immune evasion of S. aureus relies on various mechanisms to survive within the bone niche, including the secretion of leukotoxins such as Panton-Valentine leukocidin (PVL). PVL is a pore-forming toxin exhibiting selective human tropism for C5a receptors (C5aR1 and C5aR2) and CD45 on neutrophils, monocytes, and macrophages. PVL is an important virulence determinant in lung, skin and soft tissue infections. The involvement of PVL in S. aureus pathogenesis during bone infections has not been studied extensively yet. To investigate this, humanized BALB/c Rag2-/-Il2rg-/-SirpaNODFlk2-/- (huBRGSF) mice were subjected to transtibial implant-associated osteomyelitis with community-acquired methicillin-resistant S. aureus (CA-MRSA) USA300 wild type strain (WT), an isogenic mutant lacking lukF/S-PV (Δpvl), or complemented mutant (Δpvl+pvl). Three days post-surgery, Δpvl-infected huBRGSF mice had a less severe infection compared to WT-infected animals as characterized by 1) improved clinical outcomes, 2) lower ex vivo bacterial bone burden, 3) absence of staphylococcal abscess communities (SACs) in their bone marrow, and 4) compromised MRSA dissemination to internal organs (liver, kidney, spleen, heart). Interestingly, Δpvl-infected huBRGSF mice had fewer human myeloid cells, neutrophils, and HLA-DR+ monocytes in the bone niche compared to WT-infected animals. Expectedly, a smaller fraction of human myeloid cells were apoptotic in the Δpvl-infected huBRGSF animals. Taken together, our study highlights the pivotal role of PVL during acute implant-associated osteomyelitis in humanized mice.

Evolutionary dynamics of the novel ST22-PT methicillin-resistant Staphylococcus aureus clone co-harbouring Panton-Valentine leucocidin and duplicated toxic shock syndrome toxin 1 genes.

OBJECTIVES: Globally, the isolation of community-associated methicillin-resistant Staphylococcus aureus (MRSA) harbouring both the Panton-Valentine leucocidin (PVL) and toxic shock syndrome toxin 1 (TSST-1) genes is rare. However, we encountered an outbreak of the ST22-PT clone exhibiting this phenotype in Japan. Notably, the TSST-1 gene was duplicated in most of the strains. This study aimed to elucidate the mechanisms underlying this gene duplication. METHODS: A total of 90 MRSA isolates were collected from the skin of outpatients in Fukuoka City, Japan, between 2017 and 2019. Whole-genome sequencing was performed on MRSA strains that were PVL and TSST-1 positive. RESULTS: A total of 43 (47.8%) strains produced TSST-1, 20 (22.2%) produced PVL, and 16 (17.8%) produced both. Fifteen isolates were classified as ST22/SCCmec type IVa (ST22-PT clone) and one as ST1/SCCmec type V (ST1-PT clone). Three distinct ST22-PT clones were identified: Fukuoka clone I (one PVL gene and one TSST-1 gene), Fukuoka clone II (addition of a TSST-1 gene to Fukuoka clone I), and Fukuoka clone III (marked by a chromosomal inversion in a large region from Fukuoka clone II). DISCUSSION: Fukuoka clone I may have integrated a novel pathogenicity island bearing the TSST-1 gene, leading to the emergence of Fukuoka clone II with a duplicated TSST-1 gene. This duplication subsequently instigated a chromosomal inversion in a large region owing to the homologous sequence surrounding TSST-1, giving rise to Fukuoka clone III. These findings provide crucial insights into the genetic evolution of MRSA.