Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains.

Staphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

During the Early Stages of Staphylococcus aureus Biofilm Formation, Induced Neutrophil Extracellular Traps Are Degraded by Autologous Thermonuclease.

Staphylococcus aureus extracellular DNA (eDNA) plays a crucial role in the structural stability of biofilms during bacterial colonization; on the contrary, host immune responses can be induced by bacterial eDNA. Previously, we observed production of S. aureus thermonuclease during the early stages of biofilm formation in a mammalian cell culture medium. Using a fluorescence resonance energy transfer (FRET)-based assay, we detected thermonuclease activity of S. aureus biofilms grown in Iscove's modified Dulbecco's medium (IMDM) earlier than that of widely studied biofilms grown in tryptic soy broth (TSB). The thermonuclease found was Nuc1, confirmed by mass spectrometry and competitive Luminex assay. These results indicate that biofilm development in IMDM may not rely on eDNA for structural stability. A bacterial viability assay in combination with wheat germ agglutinin (WGA) staining confirmed the accumulation of dead cells and eDNA in biofilms grown in TSB. However, in biofilms grown in IMDM, minimal amounts of eDNA were found; instead, polysaccharide intercellular adhesin (PIA) was detected. To investigate if this early production of thermonuclease plays a role in immune modulation by biofilm, we studied the effect of thermonuclease on human neutrophil extracellular trap (NET) formation using a nuc knockout and complemented strain. We confirmed that thermonuclease produced by early-stage biofilms grown in IMDM degraded biofilm-induced NETs. Additionally, neither the presence of biofilms nor thermonuclease stimulated an increase in reactive oxygen species (ROS) production by neutrophils. Our findings indicated that S. aureus, during the early stages of biofilm formation, actively evades the host immune responses by producing thermonuclease.

Production of Staphylococcal Complement Inhibitor (SCIN) and Other Immune Modulators during the Early Stages of Staphylococcus aureus Biofilm Formation in a Mammalian Cell Culture Medium.

Immune modulators are known to be produced by matured biofilms and during different stages of planktonic growth of Staphylococcus aureus Little is known about immune modulator production during the early stages of biofilm formation, thus raising the following question: how does S. aureus protect itself from the innate immune responses at these stages? Therefore, we determined the production of the following immune modulators: chemotaxis inhibitory protein of staphylococci (CHIPS); staphylococcal complement inhibitor (SCIN); formyl peptide receptor-like 1 inhibitor; gamma-hemolysin component B; leukocidins D, E, and S; staphylococcal superantigen-like proteins 1, 3, 5, and 9; and staphylococcal enterotoxin A. Production was determined during in vitro biofilm formation in Iscove's modified Dulbecco's medium at different time points using a competitive Luminex assay and mass spectrometry. Both methods demonstrated the production of the immune modulators SCIN and CHIPS during the early stages of biofilm formation. The green fluorescence protein promoter fusion technology confirmed scn (SCIN) and, to a lesser extent, chp (CHIPS) transcription during the early stages of biofilm formation. Furthermore, we found that SCIN could inhibit human complement activation induced by early biofilms, indicating that S. aureus is able to modulate the innate immune system already during the early stages of biofilm formation in vitro These results form a stepping stone toward elucidating the role of immune modulators in the establishment of biofilms in vivo and present opportunities to develop preventive strategies.

Staphylococcal Protein A Is a Key Factor in Neutrophil Extracellular Traps Formation.

Staphylococcus aureus are strong inducers of neutrophil extracellular traps (NETs), a defense mechanism of neutrophils against pathogens. Our aim was to explore the role of Protein A in S. aureus-induced NETosis. We determined the Protein A production of four different S. aureus strains and found a direct relationship between the degree of NETosis induction and Protein A production: strains producing higher concentrations of Protein A evoke significantly more NETs. A S. aureus strain in which Protein A as well as a second binding protein for immunoglobulins (Sbi) have been knocked-out (ΔSpA ΔSbi) induced significantly less NETosis than the wild-type strain. NETosis induction by this knockout strain can be rescued by the addition of purified Protein A. Dead S. aureus did not induce NETosis. In conclusion, Protein A is a determinant for NETosis induction by S. aureus.

Looking to nature for a new concept in antimicrobial treatments: isoflavonoids from Cytisus striatus as antibiotic adjuvants against MRSA.

The spread of multidrug-resistant Staphylococcus aureus strains, including methicillin-resistant S. aureus (MRSA), has shortened the useful life of anti-staphylococcal drugs enormously. Two approaches can be followed to address this problem: screening various sources for new leads for antibiotics or finding ways to disable the resistance mechanisms to existing antibiotics. Plants are resistant to most microorganisms, but despite extensive efforts to identify metabolites that are responsible for this resistance, no substantial progress has been made. Plants possibly use multiple strategies to deal with microorganisms that evolved over time. For this reason, we searched for plants that could potentiate the effects of known antibiotics. From 29 plant species tested, Cytisus striatus clearly showed such an activity and an NMR-based metabolomics study allowed the identification of compounds from the plant extracts that could act as antibiotic adjuvants. Isoflavonoids were found to potentiate the effect of ciprofloxacin and erythromycin against MRSA strains. For the structure-activity relationship (SAR), 22 isoflavonoids were assessed as antibiotic adjuvants. This study reveals a clear synergy between isoflavonoids and the tested antibiotics, showing their great potential for applications in the clinical therapy of infections with antibiotic-resistant microorganisms such as MRSA.

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review.

BACKGROUND: Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing. METHODS: In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging. RESULTS: PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis. CONCLUSION: Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

Risk Factors of Typhoid Infection in the Indonesian Archipelago.

BACKGROUND: Knowledge of risk factors and their relative importance in different settings is essential to develop effective health education material for the prevention of typhoid. In this study, we examine the effect of household level and individual behavioural risk factors on the risk of typhoid in three Indonesian islands (Sulawesi, Kalimantan and Papua) in the Eastern Indonesian archipelago encompassing rural, peri-urban and urban areas. METHODS: We enrolled 933 patients above 10 years of age in a health facility-based case-control study between June 2010 and June 2011. Individuals suspected of typhoid were tested using the typhoid IgM lateral flow assay for the serodiagnosis of typhoid fever followed by blood culture testing. Cases and controls were defined post-recruitment: cases were individuals with a culture or serology positive result (n = 449); controls were individuals negative to both serology and culture, with or without a diagnosis other than typhoid (n = 484). Logistic regression was used to examine the effect of household level and individual level behavioural risk factors and we calculated the population attributable fraction (PAF) of removing each risk significant independent behavioural risk factor. RESULTS: Washing hands at critical moments of the day and washing hands with soap were strong independent protective factors for typhoid (OR = 0.38 95% CI 0.25 to 0.58 for each unit increase in hand washing frequency score with values between 0 = Never and 3 = Always; OR = 3.16 95% CI = 2.09 to 4.79 comparing washing hands with soap sometimes/never vs. often). These effects were independent of levels of access to water and sanitation. Up to two thirds of cases could be prevented by compliance to these practices (hand washing PAF = 66.8 95% CI 61.4 to 71.5; use of soap PAF = 61.9 95%CI 56.7 to 66.5). Eating food out in food stalls or restaurant was an important risk factor (OR = 6.9 95%CI 4.41 to 10.8 for every unit increase in frequency score). CONCLUSIONS: Major gains could potentially be achieved in reducing the incidence of typhoid by ensuring adherence to adequate hand-washing practices alone. This confirms that there is a pivotal role for 'software' related interventions to encourage behavior change and create demand for goods and services, alongside development of water and sanitation infrastructure.

Sccmec type II gene is common among clinical isolates of methicillin-resistant Staphylococcus aureus in Jakarta, Indonesia.

BACKGROUND: Community Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) is a strain of MRSA that can cause infections in patients in the community, in which these patients had no previous risk factors for MRSA infection and the patient received 72 hours prior to infection when admitted to hospital. This study aims to determine and compare the characteristics of epidemiological, clinical, and molecular biology of CA-MRSA with HA-MRSA. METHODS: A total of 11 clinical strains of Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Stapylococcus aureus (MSSA) were collected from 2 hospitals in Jakarta, Indonesia in 2012. SCCmec typing was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF,fruA, and fruB) associated with vancomycin resistance was examined by simple PCR analysis. RESULTS: We found three strains of community-acquired MRSA with SCCmec type II and one strain of hospital-acquired MRSA with SCCmec type IV. The other seven strains did not contain mecA genes and SCCmec. Plasmid pUB110 was found in one strain of community-acquired MRSA and two strains of hospital-acquired MRSA. vraA genes were present in 9 of the 11 strains, vraF in 4, vraG in 5, and vraR in 4. Note worthily, three quarters of strains without pUB110 contained vraR and vraF, and 70% contained vraA, whereas 60% of strains with pUB110 contained vraG. CONCLUSION: Based on these results, we should be concerned about the possibility of transition from MRSA strains sensitive to vancomycin in VISA strains of MRSA strains obtained in clinical trials. But first we need to look the existence of natural VISA or hVISA among these MRSA strains.

Multi-locus variable-number tandem repeat profiling of Salmonella enterica serovar Typhi isolates from blood cultures and gallbladder specimens from Makassar, South-Sulawesi, Indonesia.

Multi-locus variable-number tandem repeat analysis differentiated 297 Salmonella enterica serovar Typhi blood culture isolates from Makassar in 76 genotypes and a single unique S. Typhi genotype was isolated from the cholecystectomy specimens of four patients with cholelithiasis. The high diversity in S. Typhi genotypes circulating in Makassar indicates that the number of carriers could be very large, which may complicate disease prevention and control.

New flagellin gene for Salmonella enterica serovar Typhi from the East Indonesian archipelago.

Phase variation is a property unique of some Salmonella enterica serovar Typhi strains from Indonesia. Salmonella Typhi isolates from Indonesia have been described that in addition to the phase 1 Hd flagellin gene contain a second flagellin gene named z66. S. Typhi isolates from Indonesia with a mutant Hd gene named Hj have also been described. Here, we have identified another flagellin gene of S. Typhi, named Ind, showing a closest homology with the flagellin gene of Serratia marcescens. The Ind gene was detected in 21.8% of the S. Typhi isolates from the East Indonesian archipelago, all of which contained the Hd gene. The Hj gene was not detected. The z66 gene was present in 15.4% of the isolates. The presence of these "foreign" flagellin genes could be associated with an increased risk for developing severe disease.