Effects of carbon sources and temperature on the formation and structural characteristics of food-related Staphylococcus epidermidis biofilms

Biofilms are a constant concern in the food industry;understanding the effect of environmental conditions on biofilm formation is essential to develop effective control strategies.Therefore,this study was conducted to investigate biofilms formation by Staphylococcus epidermidis under various conditions.Biofilms were cultured in nutrient broth containing different carbon source concentrations(0–10 mg/mL)on polystyrene surfaces for 32 h of incubation at 37℃or 55℃,with quantification and enumeration at 8,16,24 and 32 h.S.epidermidis developed biofilms under all tested conditions;achieved the highest yield of biofilm biomass at 2.5 mg/mL for all carbon sources at 37℃.The highest efficiency of extracellular polymeric substance(EPS)molecule production occurred under glucose availability in the growth environment,with a higher yield of biomass and a significantly smaller number of metabolically active cells than under other tested conditions.A condensed ball-shaped structure was observed under the lactose condition.Meanwhile,biofilms in the presence of maltose showed mainly opaque thick rich colonies,while a compact multilayered-shaped structure was exhibited under both glucose and sucrose conditions.These results contribute to a better understanding of the biofilm formation by S.epidermidis in order to reduce contamination and recontamination in the food industry.

Teicoplanin combined with conventional vancomycin therapy for the treatment of pulmonary methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis infections

BACKGROUND Vancomycin and teicoplanin are both antibiotics that have significant antimicrobial effects on Gram-positive cocci.AIM To explore the value of teicoplanin combined with conventional(vancomycin only)anti-infective therapy for the treatment of methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis pulmonary infections.METHODS A total of 86 patients with methicillin-resistant Staphylococcus aureus or methicillin-resistant Staphylococcus epidermidis pulmonary infections,treated in our hospital between January 2018 and February 2020,were assigned to the study and control groups using a random number table method,with 43 patients in each group.The control group received conventional treatment(vancomycin),and the study group received both teicoplanin and conventional treatment.The following indicators were assessed in both groups:the time required for symptom relief,treatment effectiveness,serum levels of inflammatory factors(procalcitonin,interleukin-1β,tumor necrosis factor-α,C-reactive protein),clinical pulmonary infection scores before and after treatment,and the incidence of adverse reactions.RESULTS Patients in the study group were observed to have faster cough and expectoration resolution,white blood cell count normalization,body temperature normalization,and rales disappearance than patients in the control group(all P<0.05);the total rate of effectiveness was 93.02%in the study group,higher than the 76.74%in the control group(P<0.05).The pre-treatment serum levels of procalcitonin,interleukin-1β,tumor necrosis factor-α,and C-reactive protein as well as the clinical pulmonary infection scores were similar among the patients in both groups.However,the post-treatment serum levels of procalcitonin,interleukin-1β,tumor necrosis factor-α,and C-reactive protein as well as the clinical pulmonary infection scores were significantly lower in the study group than in the control group(P<0.05).There was no significant difference in the incidence of adverse reactions between the groups.CONCLUSION Compared with conventional(vancomycin only)therapy,teicoplanin and vancomycin combination therapy for patients with pulmonary methicillinresistant Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis infections can improve patient clinical symptoms,modulate serum inflammatory factor levels,and improve treatment efficacy,without increasing the risk of adverse reactions.

Depression of biofilm formation and antibiotic resistance by sarA disruption in Staphylococcus epidermidis

AIM： To study the effects of disruption of sarA gene on biofilm formation and antibiotic resistance of Staphylococcus epidermidis （ S. epiderrnidis）. METHODS： In order to disrupt sarA gene, the double- crossover homologous recombination was applied in S. epiderrnidis RP62A, and tetracycline resistance gene （tet） was used as the selective marker which was amplified by PCR from the pBR322 and inserted into the locus between sarA upstream and downstream, resulting in pBT2ΔsarA. By electroporation, the plasmid pBT2ΔsarA was transformed into S. epiderrnidis. Gene transcription was detected by real-time reverse transcription-PCR （RT-PCR）. Determination of biofilm was performed in 96-well flat-bottomed culture plates, and antibiotic resistance was analyzed with test tube culture by spectrophotometry at 570 nm respectively. RESULTS： A sarA disrupted strain named S. epiderrnidis RP62AΔsarA was constructed, which was completely defective in biofilm formation, while the sarA complement strain RP62AΔsarA （pHPS9sarA） restored the biofilm formation phenotype. Additionally, the knockout of sarA resulted in decreased erythromycin and kanamycin resistance of S. epiderrnidis RP62A. Compared to the original strain, S. epiderrnidis RP62AΔsarA had an increase of the sensitivity to erythromycin at 200-400 μg/mL and kanamycin at 200-800 μg/mL respectively. CONCLUSION： The knockout of sarA can result in the defect in biofilm formation and the decreased erythromycin and kanamycin resistance in S. epiderrnidis RP62A.

Staphylococcus epidermidis meningitis in combination with acute disseminated encephalomyelitis A case report

A nine-year-old girl with Staphylococcus epidermidis meningitis combined with acute disseminated encephalomyelitis （ADEM） was enrolled in the present study, and exhibited sustained fever and headache for 5 days and a single seizure episode. Meningeal irritation and disturbance of consciousness were apparent. Cerebrospinal fluid examination revealed increased protein level and pleocytosis. Cerebrospinal fluid culture demonstrated Staphylococcus epidermidis growth. The patient was treated with intravenous cefotaxime, intravenous dexamethasone, and decreasing intracranial pressure for 3 weeks. Seizures and fever symptoms improved, but disturbance of consciousness, muscle weakness, and bilateral limb paresis were significant. IgG index was elevated and oligoclonal bands were positive in the cerebrospinal fluid. Magnetic resonance imaging demonstrated high T2-weighted signals in subcortical white matter, which were consistent with ADEM. High-dose methylprednisolone for 3 days and a 6-day pulse therapy with immunoglobulins resulted in improved clinical symptoms and cerebrospinal fluid examination. The patient＇s temperature dropped to normal, and the headache disappeared. A Staphylococcus epidermidis infection associated with ADEM is uncommon in children. Results suggested that Staphylococcus epidermidis infection could be a pathogenic factor for ADEM, and ADEM is a complication of Staphylococcus epidermidis meningitis.

Effect of Terminalia chebula Retz. extraction with water on Staphylococcus epidermidis activity and its biofilm formation

Background:Based on modern pharmacological studies,Terminalia chebula Retz.exhibits antimicrobial activity against a variety of bacteria.Previously,we found Terminalia chebula Retz.exhibited excellent antibacterial activity against Malassezia restricta.Methods:We determined the minimal inhibitory concentration(MIC)and minimum bactericidal concentration of Terminalia chebula Retz.extraction with water(TRW)against Staphylococcus epidermidis(including Staphylococcus epidermidis 1-15)using the microdilution method.Staphylococcus epidermidis 1(SE11),which was the most sensitive to TRW,was selected as the test bacterium for subsequent experiments.The time-kill curve of TRW on SE11 was generated using the viable count method.Further,an in vitro biofilm model of SE11 was constructed using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazolium hydroxide test,semi-quantitative crystal violet experiment,and scanning electron microscopy.The effects of TRW on the total amount of biofilm formation,the number of viable biofilm bacteria,and biofilm microstructure of SE11 were also determined using a semi-quantitative crystal violet experiment,viable count method,and scanning electron microscopy.Finally,the electrical conductivity and soluble protein content of the SE11 suspensions were determined.Results:The average MIC of TRW against SE11 was 0.75±1.09 mg/mL.TRW(1/2MIC and 2MIC)and zinc pyrithione(1/2MIC and 2MIC)had no significant effect on total biofilm inhibition in the adhesion stage(P>0.05)and the aggregation stage(P>0.05).Further,TRW(1/2MIC and MIC)and zinc pyrithione(1/2MIC and MIC)had no significant effect on viable biofilm bacteria in the adhesion stage(P>0.05)and aggregation stage(P>0.05).TRW destroyed the integrity of the SE11 cell membrane,resulting in leakage of intracellular substances.Conclusion:TRW inhibits SE11 biofilm formation and is similar to zinc pyrithione in the adhesion and aggregation stages,which provides a theoretical basis for its application in the field of antimicrobial additives.

Agent Petri Nets Framework for Modeling Staphylococcus epidermidis Biofilm Formation

This Staphylococcus epidermidis has been discovered as the most frequent germ detected during indwelling medical devices infection. This fact is well attached with the ability of this bacterium to form structured layered population known as biofilm. Inside S. epidermidis biofilm, bacterial cells present more different behavior than in their planktonic counterpart. This paper describes the thriving application of Petri net theory for modeling of interaction between different regulations actors leading S. epidermidis to switch from Planctonik to Biofilm. Indeed this biologic system is very sensible and has dangerous effect. We propose Agent Petri Nets model to describe and analyze the process of formation of Biofilm molecule. This model presents a formal framework based on Multi Agents system characteristics.

Lack of Association between fbe Gene with Adherence and Biofilm Formation in Staphylococcus epidermidis Clinical Isolates

Biofilm formation plays a major role in the pathogenesis of nosocomial infections caused by Staphylococcus epidermidis ( S.epidermidis ). It has been suggested that protein encoded by the fbe (fibrinogen binding protein) gene of S.epidermidis enhances bacterial adherence to medical devices and biofilm formation by binding to host fibrinogen (Fg). In this study, a 1.7 kb fbe gene fragment was amplified in 111 of 115 strains of S.epidermidis clinical isolates using PCR. Contrary to expectations, only 14 strains showed marginally increased adherence to Fg-coated polystyrene wells compared with BSA coated wells. Quantitative real-time PCR revealed no statistically significant difference in Fbe expression between Fg binding strains and Fg non-binding strains. Furthermore, in the presence of soluble Fg, S.epidermidis biofilm formation decreased in a dose-dependent manner. In contrast, the Staphylococcus aureus ( S.aureus ) strain Cowan Ⅰ and other 5 S.aureus clinical isolates showed a substantial increase in both adherence and biofilm formation in the presence of Fg. The results suggest that in S.epidermidis the fbe gene may not be associated with bacterial adherence and biofilm formation.

Comparison and Evaluation of Three Animal Models for Studyingthe Pathogenicity of Staphylococcus epidermidis

In order to compare and evaluate three animal models for studying the pathogenicity of Staphylococcus epidermidis strains, three experimental animal models, namely, murine intra-venous LD 50, mouse foreign body infection and rat central venous catheter (CVC) infection models were used to assess the relative virulence of two S. epidermidis strains, ATCC 12228 and 97-337. The results from three animal models were comparable, indicating S.epidermidis 97-337 was more virulent than strain ATCC 12228. The rat CVC infection model best mimicked the conditions of clinical patients with intra-venous catheters, and more information could be obtained from this model. We conclude that different in vivo models serve for different purposes, and the rat CVC infection model is most suitable for studying specific characteristics of catheter related infections caused by S. epidermidis strains.

Stress-Induced Dispersal of Staphylococcus epidermidis Biofilm Is Due to Compositional Changes in Its Biofilm Matrix

Biofilm formation is an important virulence factor of Staphylococcus epidermidis. However, little is known about the mechanisms of staphylococcal biofilm dispersal. In the present study, we investigated biofilm dispersal of the model biofilm-forming strain S. epidermidis RP62A under oligotrophic stress conditions. We found that oligotrophic stress led to rapid dispersal of pre-formed biofilms and concomitant changes in the composition of the extracellular matrix, including a decrease in poly-N-acetylglucosamine polysaccharide and an increase in proteins. Our results suggest that modifications in biofilm integrity caused by compositional changes in the biofilm matrix can induce biofilm dispersal.

The attachment of Staphylococcus epidermidis on the surface of a carbon paste electrode at various positive potentials:The effect of pH,incubation time,and solid-medium type

The attachment of Staphylococcus epidermidis to the surface of carbon paste electrode (CPE) by applying positive potentials (50 - 600 mV) with regard of various buffer pH, cultivation time and solid-medium type was studied. The attachment process was analyzed by measuring the electric current derived from the dye (amido black) adsorbed on the vacant areas of CPE after attachment of microbial cells. The pH was not identified as a single main factor affecting the attachment (p > 0.05), however further insight revealed that the potentials applied had different effects on the microbial cells attachment. Both cultivation time and solid-medium type significantly affected the microbial attachment. Generally, increase of cultivation time up to 168 h resulted in increase of adhesion. Applying potentials 300, 400 and 200 mV resulted in the highest attachment process for bacteria cultivated for 24, 48 and 168 h, respectively. S. epidermidis cultivated on the blood agar and Baird-Parker agar plates showed the higher extent of attachment.

The difference of endothelial adherence between the clinical and commensal strains of Staphylococcus epidermidis

Staphylococcus epidermidis is one of the leading pathogens of nosocomial infections. Twenty- eight strains of biofilm-negative Staphylococcus epidermidis, of which thirteen strains considered as the clinical strains were isolated from catheters, blood and urine of patients in Huashan Hospital （Shanghai, China） and fifteen strains considered as commensal strains were isolated from the skin of healthy students who had no contact with hospitals in recent half year, were investigated for the adherence to human umbilical vein endothelial cell line ECV304 and human cervical adenocarcinoma cell line Hela, respectively. Our results showed that the clinical strains were much more adhesive to both cells than commensal ones. The endothelial adherence may be a virulence factor associated with this bacterial pathogenesis.

Total alkaloids of Sophora alopecuroides-and matrine-induced reactive oxygen species impair biofilm formation of Staphylococcus epidermidis and increase bacterial susceptibility to ciprofloxacin

Objective:To investigate the mechanism by which total alkaloids of Sophora alopecuroides(TASA)and matrine(MT)impair biofilm to increase the susceptibility of Staphylococcus epidermidis(S.epidermidis)to ciprofloxacin.Methods:The minimum biofilm inhibitory concentration(mBIC)was determined using a 2-fold dilution method.Structure of biofilm of S.epidermidis was examined by Confocal Laser Scanning Microscope(CLSM).The cellular reactive oxygen species(ROS)was determined using a DCFH-DA assay.The key factors related to the regulation of ROS were accessed using respective kits.Results:TASA and MT were more beneficial to impair biofilm of S.epidermidis than ciprofloxacin(CIP)(P<0.05).TASA and MT were not easily developed resistance to biofilm-producing S.epidermidis.The mBIC of CIP decreased by 2-6-fold following the treatment of sub-biofilm inhibitory concentration(sub-BIC)TASA and MT,whereas the mBIC of CIP increased by 2-fold following a treatment of sub-BIC CIP from the first to sixth generations.TASA and MT can improve the production of ROS in biofilmproducing S.epidermidis.The ROS content was decreased 23%-33%following the treatment of submBIC CIP,whereas ROS content increased 7%-24%following treatment with TASA+CIP and MT+CIP combination from the first to sixth generations.Nitric oxide(NO)as a ROS,which was consistent with the previously confirmed relationship between ROS and drug resistance.Related regulatory factorssuperoxide dismutase(SOD)and glutathione peroxidase(GSH)could synergistically maintain the redox balance in vivo.Conclusion:TASA and MT enhanced reactive oxygen species to restore the susceptibility of S.epidermidis to ciprofloxacin.

A new b/aLEN-17 gene in a clinical isolate of Staphylococcus epidermidis in Shanghai, China

The introduction of the antibiotics into clinical practice has significantly reduced the mortality of infectious diseases. Although chromosomally mediated β-1actamase is natural in many genera of bacteria, the intensive use of antibiotics is the main cause for the increasing emergence of new β-1actamases. So far, more than 340 β-lactamases have been identified,1 among which, more than 200 are extended-spectrum β-lactamases （ESBLs）.2 The most prevalent β-lactamases are class A enzymes, including SHV and TEM. Genes encoding these enzymes generally located in large transferable plasmids. The dissemination of these plasmids attributes to the increasing incidence and spread of v-lactam resistance. It is important to investigate the prevalence and allelic distribution of genes encoding β-lactamase in the bacterial population in order to prevent the emergence of ESBLs in those bacteria and the spread of ESBLs in the clinical setting.

Structural insights into the CRISPR-Cas-associated ribonuclease activity of Staphylococcus epidermidis Csm3 and Csm6

The Clustered Regularly Interspaced Short _Palindromic Repeats （CRISPR）-CRISPR-associated （Cas） system is an adaptive immune system in bacteria and archaea that resists exogenous invasion through nucleic acid-mediated cleavage. In the type III-A system, the Csm complex contains five effectors and a CRISPR RNA, which edits both single stranded RNA and double stranded DNA. It has recently been demonstrated that cyclic oligoadenylates （cOAs）, which are synthesized by the Csm complex, act as second messengers that bind and activate Csm6. Here, we report the crystal structures of Staphylococcus epiderrnidis Csm3 （SeCsm3） and an N-terminally truncated Csm6 （SeCsm6AN） at 2.26 and 2.0 A, respectively. The structure of SeCsm3 highly resembled previously reported Csm3 structures from other species; however, it provided novel observations allowing further enzyme characterization. The homodimeric SeCsm6AN folds into a compact structure. The dimerization of the HEPN domain leads to the formation of the ribonuclease active site, which is consistent with the reported Csm6 structures. Altogether, our studies provide a struc- tural view of the ribonuclease activity mediated by Csm3 and Csm6 of the type III-A CRISPR-Cas system.

Penetration of erythromycin through Staphylococcus epidermidis biofilm

Background The catheter related infection caused by Staphylococcus epidermidis biofilm is increasing and difficult to treat by antimicrobial chemotherapy. The properties of biofilms that give rise to antibiotic resistance are only partially understood. This study aimed to elucidate the penetration of erythromycin through Staphylococcus epidermidis biofilm. Methods The penetration ratio of erythromycin through Staphylococcus epidermidis biofilms of 1457, 1457-msrA, and wild isolate $68 was detected by biofilm penetration model at different time points according to the standard regression curve. The RNA/DNA ratio and the cell density within the biofilms were observed by confocal laser microscope and transmission electromicroscope, respectively. Results The penetration ratios of erythromycin through the biofilms of 1457, 1457-msrA, and $68 after cultivation for 36 hours were 0.93, 0.55 and 0.4, respectively. The erythromycin penetration ratio through 1457 biofilm （0.58 after 8 hours） was higher than that through the other two （0.499 and 0.31 after 24 hours）. Lower growth rate of the cells in biofilm was shown, with reduction of RNA/DNA proportion observed by confocal laser microscope through acridine orange stain. Compared with the control group observed by transmission electrmicroscope, the cell density of biofilm air face was lower than that of agar face, with more cell debris. Conclusions Erythromycin could penetrate to the Staphylococcus epidermidis biofilm, but could not kill the cells thoroughly. The lower growth rate of the cells within biofilm could help decreasing the erythromycin susceptibility.

Antimicrobial Activity of Some Commercial Toothpastes and Antibiotics on Two Oral Pathogenic Bacteria—An in-Vitro Study

Oral health problems such as periodontal diseases, dental caries, and endodontic infections have a significant negative impact on oral health and impose a substantial financial burden on the global population. The prevalence of these issues is increasing due to the buildup of bacterial plaque and the growing resistance of bacteria to antimicrobial treatments. The aims of this study to evaluate the anti-bacterial activity of four types of antibiotics (Amoxicillin, Augmentin, Azithromycin and Metronidazole) and four types of toothpastes (Sensodyne, ipana, denta and cariax Gingival Kin) on two oral pathogenic bacteria (Streptococcus mutans and Staphylococcus epidermidis). Bacterial samples of previously isolated Streptococcus mutans and Staphylococcusepidermidis were used as test organisms and the Kirby-Bauer disc diffusion method was employed to assess the antibacterial efficacy of various antibiotics and evaluate the impact of different toothpastes using a filter paper disc agar measurement technique. Each filter disc was saturated with toothpaste solution in a test tube for approximately 30 to 40 seconds, after which they were placed on Mueller-Hinton broth bacterial cultures in petri dishes. These Petri dishes were then incubated at 37°C for 24 hours, and the clear zone’s diameter (inhibition zone in mm) was subsequently measured and the results were recorded. The results demonstrated that Sensodyne toothpaste and Metronidazole antibiotic were ineffective against both types of bacteria, while Augmentin and Amoxicillin were effective by high diameter inhibition zones of growth against S. mutans and Azithromycine against S. epidermidis. Also Ipana, Denta, and Cariax Gingival Kin toothpastes exhibited a moderate effect against the two bacteria. This study suggests that certain antibiotics and toothpastes can effectively inhibit the growth of harmful oral bacteria, but not all of them are effective.

Influence of several uropathogenic microorganisms on human sperm motility parameters in vitro

Aim: The effects of certain uropathogenic microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus, Staphylococcus epidermidis and Mycobacterium tuberculosis) on human sperm motility characteristics were studied in vitro. Methods: In 10 healthy fertile men, ejaculates were aseptically obtained by masturbation and With a swim-up technique, a sperm suspension of high motility and purity was obtained. Several uropathogenic bacteria were obtained from outpatients with genitourinary tract infections. The sperm suspension was incubated with the pathogens at a bacteria: sperm ratio of 50:1 at 37℃. The sperm mobility parameters were estimated with a computerassisted sperm analyzer (CASA) provided with a multiple-exposure photography system (Madi Corp., Zhejiang, China). Measurements were carried out at 0, 2 and 4 hours of incubation. Results: Staphylococcus aureus significantly decreased the sperm motility and viability, but Staphylococcus epidermidis, Mycobacterium tuberculosis and Neisseria gonorrhoeae did not. Conclusion: Staphylococcus aureus has an inhibitory effect on human sperm motility in vitro.

Coagulase-negative Staphylococci in Conjunctivitis and Blepharitis

Coagulase-negative staphylococcus(C-NS)are regarded as normal flora of the lids and conjunctiva.The ability of these organisms to cause conjunctivitis and blepharitis can be overlooked or disregarded.To elucidate the role of individual C-NS species in these eye diseases we compared Staphylococcus sp.isolated from the conjunctiva and lids of 50 healthy volunteers with 248 strains of Staphylococcus isolated from patients with staphylococcal conjunctivitis or blepharitis.S.epidermidis was the most frequent species isolated from the conjunctiva and lids of both groups.S.aureus was isolated only from infected patients.No individual C-NS species was found to be significantly associated with eye disease,but the colony count of C-NS after isolation was a useful indicator of conjunctivitis and blepharitis.The ability of Staphylococcus to ferment mannitol or mannose was associated with isolates only from infected patients.

An Impact of Different Silicone Breast Implants on the Bacterial Attachment and Growth

Bacterial biofilms have been implicated with breast implant complications including capsular contracture, double-capsule formation, and breast implant-associated anaplastic large cell lymphoma. However, the relationship between implant surface texture and microbial biofilm formation is insufficiently evaluated. In the present study, we examined the antimicrobial activities of different types of silicone breast implant. The growth of bacterial including Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa was compared using implants with various surface textures, including Hans Smooth, Hans SmoothFine, Allergan Smooth, Eurosilicone Smooth, Eurosilicone Texture, Sebbin Smooth, Sebbin Micro, Sebbin Texture, and Motiva Smooth. Microbial investigation revealed the increased growth of S. aureus on breast implants after 48 h, except Eurosilicone Smooth, Eurosilicone Texture, Hans SmoothFine and Sebbin Smooth material. At 48 hours, there was no major difference between the S. aureus attachment on smooth and textured implants. The results of S. epidermis attachment on the implant after 48 h showed that their growth decreased on surfaces of Motiva Smooth, Sebbin Smooth, and Eurosilicone Smooth. These results indicated that S. epidermis was unable to survive on these breast implants. Eventually, P. aeruginosa count had showed decrease of bacterial count after 48 hours compared to 24 hours in most of the implants except for Eurosilicone Texture, Sebbin Smooth and Sebbin Micro, where the count of P. aeruginosa slightly increased. This indicated that P. aeruginosa was unable to exist on the smooth surfaces. Our results show that the in vitro assay revealed no significant difference between smooth and textured surfaces and showed variable interactions and needed further molecular analysis to assess their adherence nature.