Research progress of bacterial biofilm eradication agents based on modifications of natural products / 药学学报

Bacterial biofilm widely exists in all kinds of bacteria, and is related to about 80 percent of bacterial infections. It is one of the main reasons for bacterial tolerance and resistance to existing antibiotics. Therefore, there is unmet clinical need for new anti-biofilm drugs. At present, there are three kinds of anti-biofilm agents under research, including biofilm inhibitors, biofilm dispersal agents and biofilm eradication agents. Among them, the biofilm eradication agent is unique, which can not only kill bacteria in biofilm but also eliminate biofilm as a monotherapy. Based on modifications of natural products with antibacterial activity, a variety of compounds with biofilm eradicating activities have been obtained, such as, acyldepsipeptides, pyrrolomycins, halogenated phenazines and halogenated 8-hydroxyquinolines. In this review, we summarize several major biofilm eradication agents above according to their structures and mechanisms.

Effect of ultrasonic microbubbles combined with gentamicin in the treatment of diabetic foot ulcer under low-frequency ultrasound / 中华超声影像学杂志

Objective:To investigate the effect of microbubbles combined with gentamicin on the clearance of bacterial biofilms and the healing of diabetic foot ulcers under low-frequency ultrasound.Methods:From July 2021 to June 2022, 27 patients with chronic diabetic foot ulcers complicated with infection were prospectively selected from the Trauma Center of the Second Affiliated Hospital of Chongqing Medical University. The patients were divided into low-frequency ultrasound + microbubbles + gentamicin ointment group, low-frequency ultrasound + microbubbles group, and gentamicin ointment group by using a random number table, with 9 patients in each group. The three groups were all treated with simple debridement by the same surgeon.Afterward, in the low-frequency ultrasound+ microbubbles+ gentamicin ointment group, the wounds covered by 4% microbubble suspension were firstly irradiated with low-intensity focused ultrasound for 5 min, and then evenly applied with gentamicin ointment. In the low-frequency ultrasound + microbubbles group, the wounds covered by 4% microbubble suspension were irradiated with low-intensity focused ultrasound for 5 min. The gentamicin ointment group was treated with gentamicin ointment evenly. The treatment lasted for 2 weeks, and secretions and tissue specimens were collected during and 2 weeks after the treatment, respectively. The general indexes of wound surface (including ulcer depth score, secretion exudation score, fresh granulation tissue growth score, and total index score), ulcer area, ulcer healing rate, as well as negative rate of secretion culture were compared among the three groups after treatment. Additionally, the structural changes in bacterial biofilms under a scanning electron microscope and colony count under a laser confocal scanning microscope were compared among the three groups after treatment.Results:No significant differences were found in the general datas among the three groups (all P>0.05). After treatment for 2 weeks, the overall general indexes showed statistically and significant differences among the three groups (all P<0.05). Each index score in the low-frequency ultrasound + microbubbles + gentamicin ointment group was lower than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). There were no significant differences in overall ulcer area among the three groups ( P>0.05). The overall ulcer healing rate presented significant differences among the three groups ( P<0.05). The healing rate in the low-frequency ultrasound + microbubbles + gentamicin ointment group was higher than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). The overall negative rates of secretion culture among the three groups were significantly different ( P<0.05), the negative rate in the low-frequency ultrasound + microbubbles + gentamicin ointment group was higher than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (all P<0.05). Scanning electron microscopy confirmed bacterial biofilm infection in the three groups before treatment. After treatment for 2 weeks, the biofilm formation in the low-frequency ultrasound + microbubbles + gentamicin ointment group reduced significantly, while the low-frequency ultrasound + microbubbles group and the gentamicin ointment group had little change compared with that before treatment. Significant differences were detected in total colony count among the three groups under the confocal microscope ( P<0.05). The colony count in the low-frequency ultrasound + microbubbles + gentamicin ointment group was lower than that in the low-frequency ultrasound + microbubbles group and the gentamicin ointment group (both P<0.05). Conclusions:Ultrasound microbubbles combined with gentamicin can clear bacterial biofilms and promote the healing of diabetic foot ulcers.

Bacterial flora of biofilm in the endotracheal tube of mechanically ventilated neonates and its relationship with ventilator-associated pneumonia / 中华传染病杂志

Objective:To explore the relationship between endotracheal tube-bacterial biofilm (ETT-BF) in mechanically ventilated neonates and ventilator-associated pneumonia (VAP).Methods:A total of 30 mechanically ventilated neonates whose mechanical ventilation time were ≥48 h in the Department of Neonatology in The Second Affiliated Hospital of Wenzhou Medical University from January 2019 to January 2020 were included.According to the indwelling time of endotracheal tube, all cases were divided into three groups including group A(two to six days), group B(seven to 14 days) and group C (over 14 days). The morphological results of ETT-BF were scanned by scanning electron microscope (SEM). The incidence of VAP, the positive rates of strains isolated from endotracheal tube surface and lower respiratory tract secretion, the detection of strains and drug resistance were analyzed. Chi-squared test were used for statistical analysis.Results:The results of SEM showed that sheet matrix could be observed on the surface of the inner cavity of endotracheal tube in three days of tracheal catheter retention, and cocci adhered to it in four days. With prolonged indwelling time of endotracheal tube, the structure of bacterial biofilm (BF) had improved.The positive rate of strains isolated from the secretion of lower respiratory tract in 30 neonates was 23.3%(7/30) and all of them were Gram-negative bacteria. There was no patient developed VAP in group A, while there were two patients with VAP in group B, and five patients with VAP in group C. The incidences of VAP in the three groups were statistically significant ( χ2=10.82, P=0.004). There was no significant difference in the positive rate of strains isolated from the surface of endotracheal tube under different indwelling time in 30 cases ( χ2=1.03, P=0.598). Among of 13 neonates in group A, there were seven strains isolated from ETT-BF, mainly Gram-positive bacteria which turned out to be mainly Gram-negative bacteria with the prolongation of endotracheal tube indwelling time. Of the seven VAP cases, strains isolated from the lower respiratory tract secretion were consistent with the strains isolated from the surface of the corresponding endotracheal tube in five cases, which were Serratia liquefaciens, Klebsiella acidogenes, Serratia marcescens, Flavobacterium meningosepticum and Stenotrophomonas maltophilia, and the drug resistance was consistent. Conclusions:The colonization bacteria of early ETT-BF may come from the upper respiratory tract, with less migration which rarely causes VAP. With the prolongation of endotracheal tube indwelling time, the incidence of VAP in neonates increases. The same pathogen can be found in the ETT-BF and lower respiratory tract secretion. The source of pathogen needs further study.

Using plant extracts and their active ingredients to inhibit bacterial biofilms / 生物工程学报

Numerous studies have reported that the resistance of biofilm bacteria to antibiotics can be up to 10-1 000 fold higher than that of planktonic bacteria. Bacterial biofilms are reported to be responsible for more than 80% of human microbial infection, posing great challenges to the healthcare sector. Many studies have reported that plant extracts and their active ingredients can inhibit the formation and development of bacterial biofilms, including reducing biofilm biomass and the number of viable bacteria in biofilms, as well as eradicating mature biofilms. This review summarized the plant extracts and their active ingredients that are inhibitory to bacterial biofilms, and analyzed the underpinning mechanisms. This review may serve as a reference for the development of plant drugs to prevent and treat biofilm infections.

Resin Nano-Ceramic Material Surface Roughness, Color Stability, and Bacterial Adhesion Evaluation After Different Scaling Methods / Rugosidade superficial, estabilidade de cor e adesão bacteriana de resina nano-cerâmica após diferentes métodos de raspagem

Objective: TThe study evaluated in vitro the changes in roughness, color stability, and bacterial count of a CAD/CAM Resin Nano-Ceramic material surface treated by various scaling procedures. Material and Methods: 70 disks (5mm diameter, 0.5 mm thickness) of Resin Nano-Ceramic (Lava ™Ultimate, 3M, ESPE) material were cemented in standardized cavities prepared in bovine teeth. A custom-made scaling apparatus of a double pan balance was used for different scaling methods, simulating standard clinical conditions. The specimens were assigned to three main groups: no scaling(C), ultrasonic scaling (U), and manual scaling (M). Each group was then divided into three subgroups according to scaling tip material; stainless steel tip (St), plastic tip (P), and titanium tip (Ti). The surface texture was analyzed quantitatively and qualitatively with a tactile profilometer and atomic force microscopy. A spectrophotometer was used for color measurement. Streptococcus mutans were counted in a colony counter. All the data were tabulated and statistically analyzed. Results: Two-way ANOVA was used to study the effect while One-way ANOVA was performed to compare between study groups. The significance level was set at p ≤ 0.05. The ultrasonic titanium tip(UTi) revealed the significant highest mean value of alterations (p < 0.001). The integrity of the material surface was altered in the form of deep scratches on the ultrasonically scaled surfaces and numerous smaller scratches on the hand-scaled surfaces. Conclusion: The plastic instrument would appear to be the instrument of choice during a routine maintenance procedure for Resin Nano-Ceramic materials (AU).

Objetivo: Este estudo avaliou in vitro as alterações na rugosidade, estabilidade de cor e quantidade de bactérias da superfície de uma resina nano-cerâmica produzida em CAD/CAM tratada por diferentes procedimentos de raspagem. Material e Métodos: 70 discos (5 mm diâmetro, 0,5 espessura) de resina nano-cerâmica (Lava ™Ultimate, 3M, ESPE) foram cimentadas em cavidades padronizadas preparadas em dentes bovinos. Um aparato customizado de raspagem com pratos duplos de balança foram usados para os diferentes métodos de raspagem, simulando uma condição clínica padronizada. Os espécimes foram distribuídos em três principais grupos: Sem raspagem (C), raspagem ultrassônica (U) e raspagem manual (M). Cada grupo foi dividido em três subgrupos de acordo com a ponta do material de raspagem; ponta de aço inoxidável (St), ponta plástica (P), ponta de titânio (Ti). A textura da superfície foi analisada qualitativamente e quantitativamente por um perfilômetro tátil e microscopia de força atômica. Um espectrofotometro foi usado para a mensuração da cor. Estreptococos mutans foram contados em um contador de colônias. Todos os dados foram tabulados e analisados estatisticamente. Resultados: Anova dois fatores foi utilizado para estudar os efeitos, enquanto ANOVA um fator foi utilizado para comparar os grupos experimentais. O nível de significância foi estabelecido em p≤ 0,05. A ponta de ultrassom de titânio (UTi) revelou o valor significante mais alto de alterações (p < 0,001). A integridade da superfície d matérias foi alterada na forma ranhuras profundas, nas superfícies raspada por ultrassom e numerosas ranhuras menores nas superfícies raspadas à mão. Conclusão: O instrumento plástico poderia apresentar-se como um instrumento de escolha durante o pocedimento de manutenção routineira para materiais de resina nano-ceramica. (AU)

Study on inhibitory effect of Ag +-loaded TiO 2 on the biofilm of Staphylococcus aureus / 中华危重病急救医学

Objective:To observe the antibacterial effect of Ag +-loaded TiO 2 (Ag -TiO 2) and Ag -TiO 2 coated endotracheal tube (ETT) on the bacterial biofilm (BF) of Staphylococcus aureus. Methods:2, 3-bis-(2-methoxy- 4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) colorimetric method was used to detect minimal inhibitory concertation (MIC) of Ag-TiO 2 for inhibition of BF of Staphylococcus aureus. The Ag -TiO 2 coated ETT were prepared, and divided into 11 mg/L, 8 mg/L, 5 mg/L, 2 mg/L and 0 mg/L ETT group, according to the concentration gradient, then impregnated in the liquid with Staphylococcus aureus at a concentration of 1.0×10 9cfu/L. The influence of antibacterial coated ETT on the formation of Staphylococcus aureus BF was determined by detecting the colonies of bacteria and BF on the ETT. Results:Ag-TiO 2 had a significant inhibitory effect on Staphylococcus aureus BF in a concentration -dependent manner, and its MIC was 10 mg/L. Ag -TiO 2 coated ETT has significant anti -Staphylococcus aureus BF effect, and the higher the concentration, the stronger the effect. The absorbance ( A) values of Ag -TiO 2 5 mg/L, 8 mg/L, 11 mg/L ETT groups were significantly lower than that in control group (0.176±0.004, 0.147±0.002, 0.094±0.002 vs. 0.267±0.045, all P < 0.05). The inhibitory rates of Ag -TiO 2 2 mg/L, 5 mg/L, 8 mg/L ETT groups were increased gradually, and 11 mg/L Ag -TiO 2 coated ETT group had the highest inhibitory rate for BF, the inhibitory rates were 6.4%, 34.1%, 44.9% and 64.8%, respectively. Conclusion:Both Ag-TiO 2 and Ag-TiO 2 coated ETT have significant inhibitory effects on Staphylococcus aureus BF.

Relationship of biofilm-forming ability of with swimming motility, twitching motility and virulence gene distribution / 浙江大学学报·医学版

To investigate the relationship of biofilm-forming ability of (PA) with swimming motility, twitching motility and virulence gene distribution. A total of 192 clinical isolates of PA were collected consecutively. Microtiter plate method was used to evaluate the ability to form biofilm. The swimming and twitching motilities were detected by plate method. Polymerase chain reaction (PCR) was used to detect virulence genes. Of the 192 PA clinical isolates, 186 (96.9%) showed biofilm-forming ability. Among them, 36 isolates showed weak biofilm-forming ability, 84 exhibited moderate biofilm-forming ability and 66 showed strong biofilm-forming ability. The diameters of the swimming ring for PA with none biofilm-forming ability, weak biofilm-forming ability, moderate biofilm-forming ability, strong biofilm-forming ability were (9.12±6.76), (18.42±7.51), (19.10±4.77) and respectively. The diameters of the twitching ring for PA in above groups were (8.38±1.50), (17.21±7.42), (18.49±5.62) and respectively. The swimming motility and twitching motility of none biofilm-forming ability group were weaker than biofilm-forming ability groups (all <0.05). Among 192 PA strains, 163 were positive (84.9%), 40 were positive (20.8%), 183 were positive (95.3%), and 189 were positive (98.4%). The positive rate of PA virulence gene , and were different in strains with different biofilm-forming abilities (<0.05). The rate of in the strong biofilm-forming ability group was lower than that in the moderate biofilm-forming ability group (=9.293, <0.01) and the weak biofilm-forming ability group (=9.997, <0.01). The rate of in the strong biofilm-forming ability group was higher than that in the weak biofilm-forming ability group (=10.803, <0.01). Most clinical isolates of PA can form biofilm. Swimming and twitching motilities are related to the formation of biofilm, but not significantly related to strength of biofilm-forming ability. The virulence genes of type Ⅲ secretion system for PA may be related to the biofilm-forming ability.

Urine turbulent shear stress system of bionic human bladder based on bacterial biofilm reactor: In vitro construction / 中国组织工程研究

BACKGROUND: The formation of bacterial biofilm on the material surface is the core problem of catheter-related urinary tract infection. Many researches have focused on the mechanism and prevention of such category of infection under static or simple hydrodynamic stimulation. The construction of dynamic model of bacterial biofilm of bladder urine flow close to real human diseases is the key to study the pathological mechanism and develop new technology of anti-biofilm infection. OBJECTIVE: To put forward the concept of turbulent flow shear stress of human bladder urine flow, construct this turbulent shear stress system based on the bacterial biofilm reactor of in vitro bionic human bladder, and explore the formation of E. coli biofilm stimulated by different stresses. METHODS: An in vitro dynamic bionic bladder urine flow model was designed. E. coli standard strain ATCC25922 was used as research object, and the medical silica gel was used as bacterial biofilm forming carrier. Four artificial urine flow stresses were simulated: hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress and pathological turbulent flow shear stress (simulated urine retention environment). A bacterial biofilm reactor loaded with turbulent flow shear stress was established. Optical density value, colony count, and biofilm surface area of bacterial biofilm suspension were detected 24, 72, 120, and 168 hours. RESULTS AND CONCLUSION: (1) Optical density value of bacterial membrane suspension: there was significant difference between different urinary stress groups and different test time points (F=110.84, 187.96, all P < 0.000 1), and there was interaction effect between time and stress (F=50.05, P < 0.000 1). From hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress, to pathological turbulent flow shear stress, the number of biofilm bacterial colonies increased. (2) Colony count of biofilm bacterial suspension smear: there was significant difference between different time (F=6.30, P=0.002 9); no difference was found between different urinary stress groups (F=1.11, P=0.400 1); and there was no interaction effect between time and stress (F=0.85, P=0.581 4). However, with the time extension of stress action, the colony count of complex stress group showed an increasing tendency, especially in the pathological turbulent shear stress. (3) Scanning electron microscopic characterization of biofilm bacteria: qualitative comparison between each group and different time points showed that the formation of bacterial biofilm was different from sparse fragments, lumps to large lumps. There were significant differences in the bacterial biofilm surface area between different urinary stress groups and at different times (F=505.72, 1 201.84, all P < 0.000 1), and there was interaction effect between time and stress (F=78.14, P < 0.000 1). From hydrostatic pressure, constant turbulent flow shear stress, physiological turbulent flow shear stress, to pathological turbulent flow shear stress, the biofilm formation increased significantly. (4) The results showed that this turbulent flow shear stress of human bladder urine flow can obviously stimulate E. coli biofilm formation in vitro. Its functional changes and pathogenic mechanism need to be further explored.

Pathogenic distribution and drug resistance of bacterial biofilm on ureteral stent / 中国组织工程研究

BACKGROUND: Ureteral stents have been extensively applied In the stenosis In the conjunction of the renal pelvis and ureter, the reconstruction of in situ urine flow, ureter or nephroscope lithotripsy, renal transplant, and tumors. However, the long-term retention of ureteral stents can induce catheter-associated urinary tract Infection complications. OBJECTIVE: To Investigate the morphological characteristics of bacterial blofilm on ureteral stent, and to analyze the features of pathogenic distribution and antimicrobial drug resistance to bacterial biofilm. METHODS: Specimens of ureteral stent were collected from 127 patients at Yongchuan Hospital, Chongqing Medical University between January and December 2016. The morphological characteristics of bacterial blofilm on the stent were observed under scanning electron microscope. Each specimen was divided Into three parts (renal pelvis, ureter and bladder) for screening biofilm-forming bacteria strains separately by Congo red medium. The urine was bacterially cultured. Drug susceptibility test was done with the collected biofilm-forming bacteria strains. The study was approved by the Ethics Committee of Yongchuan Hospital, Chongqing Medical University (approval No. 201422). RESULTS AND CONCLUSION: (1) Bacterial blofilm was observed on the surface of ureteral stents at 7,15 and 30 days of retention, with various numbers of inflammatory attachments or crystals. Bacteria on the bacterial biofilm were embraced by large amounts of fiber membranes. Patchy bacterial colonies were observed on the surface of the ureteral stent at 7 and 15 days of retention, which mainly focused on bacillus. Heap-shaped bacterial colonies were found on the surface of ureteral stents that were retained for 30 days, which mainly were bacillus and coccus. (2) A total of 106 bacterial blofilms were detected in the ureteral stent samples obtained from 127 patients. The positive rate was 83.5%, in which the bladder section had the highest positive rate, followed by the renal pelvis section and ureter section. There were 25 copies of positive urine culture, and the positive rate was 19.7%. The strains obtained from the bacterial biofilm on each section of the ureteral stents were significantly higher than that from the urine bacteria culture (P < 0.05). (3) A total of 227 strains were detected from 106 positive samples. Among these samples, the number of Gram-negative strains was significantly higher than that of Gram-positive strains (P < 0.05). Among culture bacteria of the bacterial biofilm on the ureteral stent and urine culture bacteria, colibacillus, pseudomonas aeruginosa, enterococcus faecalis and enterococcus faecium were the most common. (4) The biofilm-forming bacteria on the ureteral stent had a high drug resistance. (5) In summary, bacterial blofilm may be the important reason for catheter-associated urinary tract infection.

Current progress in antimicrobial peptides against bacterial biofilms / 生物工程学报

Microbial biofilm, a consortium of microbial cells protected by a self-produced polymer matrix, is considered as one main cause of current bacterial drug resistance. As a new type of antimicrobial agents, antimicrobial peptides provide a new strategy for the treatment of antibiotic resistant bacteria biofilm infections. Antimicrobial peptides have shown unique advantages in preventing microbial colonization of surfaces, killing bacteria in biofilms or disrupting the mature biofilm structure. This review systemically analyzes published data in the recent 30 years to summarize the possible anti-biofilm mechanisms of antimicrobial peptides. We hope that this review can provide reference for the treatment of infectious diseases by pathogenic microbial biofilm.

Study of Fusobacterium nucleatum -related bacterial biofilm promoting M2 polarization of macrophages and chemoresistance in colon cancer / 上海交通大学学报（医学版）

Objective: To investigate whether and how Fusobacterium nucleatum-related bacterial biofilm modulates the infiltration of tumor-associated macrophages into tumor microenvironment and the response to chemotherapy in colon cancer patients. Methods: Both biofilm-based F.n-culture medium (BF-CM) and planktonic F.n-culture medium, (P-CM) Fusobacterium nucleatum was cultured, and the culture-medium was collected to coculture with CRC cell lines and macrophages. Quantified real time PCR( qRT-PCR) was used to measure genes related to chemoresistance, CCK8 assay was conducted to measure proliferation inhibition rate of chemicals to cancer cells, qRT-PCR was used to measure the expression of genes related to macrophage polarization. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) were conducted to evaluate existence of bacterial biofilm and infiltration of macrophages in tumor tissues of colon cancer. Results: Expression of chemoresistance-related genes(e.g. MDR1/Abcb1a/ Abcb1b) were higher in BF-CM treated CRC cells than those in P-CM treated cells. CRC cell inhibition level via LOHP/5-FU were reduced with F.n culture medium(metabolites) co-culture, and much lower in BF-CM group. Expression of M2-polarization markers were also higher after BF-CM treated macrophages than P-CM. Biofilm positivity was higher in recurrent(confirmed by PET-CT, CT, or colonoscopy) colon cancer patients with post-resection adjuvant chemotherapy, than that in non-recurrent ones; correlated with infiltration rate of M2 macrophages. Conclusion: Fusobacterium nucleatumrelated bacterial biofilm can induce M2-polarization of intratumor macrophages and could promote chemoresistance to chemicals in CRC cells, which may contribute to prognosis of colon cancer patients.

Chitosan membrane type under the negative pressure therapy for infected wounds multi-resistant bacteria flora changes of examination / 中华整形外科杂志

Objective@#To evaluate the outcome of negative pressure closed drainage with chitosan membrane in the treatment of multiple drug-resistant bacterial infections.@*Methods@#From January 2015 to December 2017, 108 patients with skin ulcer wound complicated by multiple drug-resistant bacterial infection were admitted in the department of burn and plastic surgery, Qingdao Jiaozhou Central Hospital. Among them, 36 patients had pressure ulcers, 40 cases had diabetic foot wounds, and 32 were traumatic skin ulcer wounds. Patients were divided into group A or group B for different treatments. In group A, besides the basic surgical dressing change, patients were treated by negative pressure closed drainage with chitosan membrane. The patients in Group B were only treated with basic surgical dressing change. The changes of wound were closely observed during the phases, and the wound bacterial culture and antimicrobial drug sensitivity test were performed regularly. The therapeutic effects of the 2 groups were compared. The changes of bacterial species of wound infection and the healing time were recorded.@*Results@#In group A, the healing time of wound infection was: pressure ulcers (14.00±1.28) days, diabetic foot wounds (13.40±1.27) days, traumatic skin ulcer wounds (12.44±1.55) days. In group B, the wound healing time was: pressure ulcers (25.17±2.73) days, diabetic foot wounds (23.85±1.73) days, traumatic skin ulcer wounds (19.81±1.94) days. The wound healing time of group A was shorter than group B. In group A, the multiple drug-resistant bacteria was replaced by non-multiple drug-resistant bacteria, or there was no pathogenic bacterial growth. The differences between the two groups was statistically significant (all P<0.05).@*Conclusions@#Additional to the basic surgical dressing change, negative pressure closed drainage with chitosan membrane could promote wound healing, when it′s associated with multiple drug-resistant bacteria infection. This method has benefits in efficient drainage, preventing the formation of bacterial biofilm and changing local microenvironment for the dominant propagation. Therefore, it could effectively control the multiple drug-resistant bacterial infections, promote wound healing and save treatment time.

In vitro activity of ceftazidime-avibactam combined with colistin against extensively drug-resistant Pseudomonas aeruginosa / 中华危重病急救医学

Objective@#To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA).@*Methods@#Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT).@*Results@#The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109).@*Conclusions@#CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.

In vitro activity of ceftazidime-avibactam combined with colistin against extensively drug-resistant Pseudomonas aeruginosa / 中华危重病急救医学

Objective To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Methods Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). Results The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). Conclusions CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.

In vitro activity of ceftazidime-avibactam combined with colistin against extensively drug-resistant Pseudomonas aeruginosa / 中华危重病急救医学

Objective To evaluate the in vitro activity of ceftazidime-avibactam (CAZ-AVI) alone or in combination with colistin (COL) against clinically isolated extensively drug-resistant Pseudomonas aeruginosa (XDR-PA). Methods Minimum inhibitory concentration (MIC) of 16 clinical XDR-PA isolates was determined by broth dilution method and chessboard design when CAZ-AVI and COL were used alone or in combination, then the combined inhibitory concentration index (FICI) was calculated. Class A [Klebsiella pneumoniae carbapenemase β-lactamase (blaKPC), Guiana extended-spectrum β-lactamase (blaGES)], Class B [imipenemase β-lactamase (blaIMP), Verona-Integronmetallo β-lactamase (blaVIM), New Delhi metallo β-lactamase (blaNDM), German imipenemase β-lactamase (blaGIM), Sao Paulo metallo -β- lactamase (blaSPM)], Class C [AmpC β-lactamase (blaAmpC)], Class D [oxacillinase β-lactamase (blaOXA)] β- lactamase-related resistance genes were detected by polymerase chain reaction. Drug-resistant mutation frequencies of each strain were determined on a drug-containing plate. The time kill curves of three XDR-PA were plotted by colony counting method. A biofilm model was established in vitro, and the synergistic effect of CAZ-AVI and COL on biofilm inhibition was detected by methythiazolyl tetrazolium assay (MTT). Results The MICs of 16 XDR-PA for CAZ-AVI ranged from 1 mg/L to 128 mg/L, and three of the isolates showed resistance (MIC > 8 mg/L). The FICI range of CAZ-AVI combined with COL was 0.312-1.000. Four isolates were synergistic, while the other 12 isolates were additive. Three isolates resistant to CAZ-AVI contained Class B resistance genes such as blaIMP and blaVIM, while 13 susceptible isolates carried resistance genes belonging to Class A, C or D. The logarithm values of mutation frequencies of drug resistance in CAZ-AVI group, COL group and combination group were -4.81±0.88, -7.06±0.69 and -9.70 (-9.78, -9.53), respectively. There were significant differences among the three groups (H = 33.601, P < 0.001), and between every two groups (adjusted P < 0.05). In time kill curves, the phytoplankton load of three XDR-PA decreased more than 6 log CFU/L when these two drugs were used together, and number of PA1819 planktonic bacteria decreased more than 5.1 log CFU/L compared with monotherapy group. Viable quantity in biofilm (A490) of normal saline group, CAZ-AVI group, COL group and CAZ-AVI-COL group were 0.665±0.068, 0.540±0.072, 0.494±0.642 and 0.317±0.080, respectively. There was significant difference between the other two groups (all P < 0.001), except for that between CAZ-AVI group and COL group (P = 0.109). Conclusions CAZ-AVI combined with COL can effectively improve the bactericidal effect of each drug alone on XDR-PA. The regimen can also reduce the production of drug-resistant bacteria and inhibit the formation of biofilm. Therefore, it is a potential treatment for XDR-PA infection.

The inhibition of accessory gene regulator C specific binding peptides on biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride in vitro / 中国修复重建外科杂志

Objective: To investigate the effect of accessory gene regulator C (agr C) specific binding peptides (named N1) on the biofilm formation of Staphylococcus epidermidis on the surface of polyvinyl chloride (PVC) materials in vitro. Methods: Firstly, the two strains (ATCC35984, ATCC12228) were cultured with N1 at concentrations of 100, 200, 400, 800, and 1 600 μg/mL, respectively. The control group was cultured with agrC specific binding unrelated peptides (named N0) at the same concentrations and the absorbance ( A) value was measured after 24 hours to determine the optimal bacteriostatic concentration of N1. The two strains were cultured with N1 and N0 of the optimal concentration, respectively. The A values were measured at 6, 12, 18, 24, 30, and 48 hours to observe the effect of N1 on the biofilm formation ability of Staphylococcus epidermidis. On this basis, the surface structure of the biofilm on the surface of PVC material was observed by scanning electron microscopy after 6, 12, 18, 24, and 30 hours of incubation with PVC material sheet. The thickness of the biofilm was observed by laser confocal microscopy after 6, 12, 18, and 24 hours of incubation with ATCC35984 strain. Results: The optimal bacteriostatic concentration of N1 was 800 μg/mL. ATCC 12228 strain did not form obvious biofilm after being cultured with N1 and N0. When ATCC35984 strain was cultured with N1 and N0 for 12 hours, the difference in biofilm formation ability between groups N1 and N0 was statistically significant ( P0.05). Scanning electron microscopy examination showed that mature biofilm structure was observed in ATCC35984 strain and was not observed in ATCC12228 strain. Laser confocal microscopy observation showed that the number of bacteria in the group N1 was significantly lower than that in the group N0 at 12 hours, and the most of bacteria were dead bacteria. There was no significant difference in the number of bacteria at 6, 18, and 24 hours, and the most of them were live bacteria. The biofilm thickness of group N1 was significantly lower than that of group N0 at 12 and 18 hours ( P<0.05). Conclusion: The intensity of N1 inhibiting the formation of Staphylococcus epidermidis biofilm is dose-dependent. During the aggregation period, N1 can inhibit the biofilm formation by hindering the bacterial growth and aggregation. The inhibition effect on mature biofilm is not obvious.

Effects of various prophylactic procedures on titanium surfaces and biofilm formation

PURPOSE: The aim of this study was to evaluate the effects of various prophylactic treatments of titanium implants on bacterial biofilm formation, correlating surface modifications with the biofilms produced by Pseudomonas aeruginosa PAO1, Staphylococcus aureus, and bacteria isolated from saliva. METHODS: Pure titanium disks were treated with various prophylactic procedures, and atomic force microscopy (AFM) was used to determine the degree to which surface roughness was modified. To evaluate antibiofilm activity, we used P. aeruginosa PAO1, S. aureus, and saliva-isolated Streptococcus spp., Bacteroides fragilis, and Staphylococcus epidermidis. RESULTS: AFM showed that the surface roughness increased after using the air-polishing device and ultrasonic scaler, while a significant reduction was observed after using a curette or polishing with Detartrine ZTM (DZ) abrasive paste. In addition, we only observed a significant (P < 0.01) reduction in biofilm formation on the DZ-treated implant surfaces. CONCLUSION: In this study, both AFM and antibiofilm analyses indicated that using DZ abrasive paste could be considered as the prophylactic procedure of choice for managing peri-implant lesions and for therapy-resistant cases of periodontitis.

Efficacy of a Kalanchoe gastonis-bonnieri extract to control bacterial biofilms and dental calculus in dogs / Eficácia do extrato de Kalanchoe gastonis-bonnieri no controle do biofilme bacteriano e cálculo dentário em cães

An aqueous leaf extract of the medicinal species Kalanchoe gastonis-bonnieri (here denominated KGB) has been found to be effective as an antimicrobial agent against canine oral cavity bacteria in in vitro assays. In this study, we investigated the effect of topic oral administration of KGB on the development of dental biofilm in Beagle dogs. The experiments were performed with an experimental group (0.2% of KGB extract), a negative control group (0.9% of saline solution) and a positive control group (0.12% chlorhexidine). Each treatment was sprayed into the oral cavity daily for 28 days. Thirty Beagle dogs with similar characteristics and kept under the same management and diet were used. The measurement of dental plaque and calculus was performed using a computerized analytical method. The phenolic profile of KGB extract was analyzed by HPLC-DAD. KGB extract at 0.2% showed efficacy in controlling the formation of plaque compared to the negative control group, and dental calculus in relation to the negative and positive control groups. A significant difference was observed among these three groups. Peaks attributed to flavonoids and phenolic acids were identified in the HPLC-DAD chromatogram of the KGB extract. The presence of these substances could be related to the activity observed. Our findings demonstrate that treatment with KGB is effective in controlling periodontal disease in dogs, providing new insights into the medicinal properties of this plant. KGB extract has a potential use as a supplemental agent in pharmaceutical products for the prevention of periodontal disease.(AU)

Um extrato aquoso de folhas da espécie medicinal Kalanchoe gastonis-bonnieri (aqui denominado como KGB) foi efetivo como um agente antimicrobiano contra as bactérias da cavidade oral de cães em testes in vitro. Neste estudo, investigou-se o efeito da administração oral tópica de KGB sobre o desenvolvimento do biofilme dental em cães da raça Beagle. Os experimentos foram realizados com um grupo experimental (0,2% de extrato de KGB), um grupo controle negativo (0,9% de solução salina) e um grupo controle positivo (0,12% de gluconato de clorexidina). Cada tratamento foi aplicado no interior da cavidade oral diariamente durante 28 dias. Foram utilizados trinta cães da raça Beagle com características semelhantes e mantidos sob o mesmo manejo e dieta. A medição da placa bacteriana e cálculo dentários foi realizada utilizando-se um método de análise computadorizada. O perfil fenólico do extrato de KGB foi analisado por HPLC-DAD. O extrato de KGB a 0,2% mostrou eficácia no controle da formação de placa bacteriana em comparação com o grupo controle negativo, e de cálculo dentário em relação aos grupos controle negativo e positivo. Uma diferença significativa foi observada entre esses três grupos. Picos atribuídos a flavonoides e ácidos fenólicos foram identificados no cromatograma de HPLC-DAD do extrato de KGB. A presença de tais substâncias pode estar relacionada com a atividade observada. Os resultados demonstram que o tratamento com KGB é eficaz no controle da doença periodontal em cães, fornecendo novas perspectivas sobre as propriedades medicinais desta planta. O extrato de KGB tem uma utilização potencial como um agente suplementar em produtos farmacêuticos para a prevenção da doença periodontal.(AU)

Study on in vitro drug susceptibility test of biomembrane-producing Klebsiella pneumoniae / 国际检验医学杂志

Objective To study the difference of in vitro drug susceptibility test of biomembrane-producing Klebsiella pneumoni-ae to provide accurate drug reference for clinical treatment.Methods The drug susceptibility test was carried out in clinically isola-ted and screened16 strains of biomembrane-producing Klebsiella pneumoniae by K-B paper diffusion method ,and the results at 24 , 48 ,72 h were recorded.Then the strains were reproduced for several generations until not producing biofilm.Then the above opera-tions were taken again.Finally ,the results of twice drug susceptibility tests were performed the contrastive analysis.Results The mm number of inhibition zone of biomembrane-producing Klebsiella pneumoniae at 24 h had statistical difference compared with that at 48 ,72 h(P<0.05).Conclusion When the in vitro drug susceptibility test of biomembrane-producing Klebsiella pneumoniae is conducted by K-B paper diffusion method ,it is suitable to report the results after 48 h ,this has a certain reference value for pre-venting this bacterium.

Study on main pharmacodynamics and underlying mechanisms of 999 Ganmaoling / 中国中药杂志

To observe synergistic effects of 999 Ganmaoling (GML) and its Chinese/Western materia medica (CMM and WMM) on pharmacodynamic action and to study underlying mechanisms, their anti-inflammatory, antipyretic effects were compared by assaying the increased capillary permeability induced by glacial acetic acid in mice, ear swelling induced by Xylene in mice, non-specific pleurisy induced by carrageenan in rats, and yeast induced fever in rats. Crystal violet (CV) and microbial activity (XTT) assay were used to evaluate the inhibition of GML and its CMM and WMM on KPN biofilm formation, and scanning electron microscopy (SEM) was applied for observing KPN biofilm morphology changes. The results showed that compared with control group, GML could reduce exudation amount of Evans-Blue and the degree of Ear swelling significantly, and CMM and WMM have no significant effects. The concentration of TNF-α and IL-1β of rat pleural effusion in GML, CMM and WMM group decreased significantly. The concentration of TNF-α, IL-1β and IL-8 in GML group, TNF-α, IL-8 in WMM group and IL-8 in CMM in rats serum decreased significantly. The body temperature in rats decreased significantly in GML and WMM group after 4-8 h of administration. CMM group showed no significant difference in rat body temperature compare with control. Compared with control group, GML (55-13.75 g•L⁻¹) could inhibit KPN biofilm formation and reduce number of viable cells in the KPN biofilm. CMM (45-22.5 g•L⁻¹) and WMM (10 g•L⁻¹) could also inhibit KPN biofilm formation and reduce number of viable cells (P<0.01). Result of SEM also showed that GML (55 g•L⁻¹) and its CMM (45 g•L⁻¹) and WMM (10 g•L⁻¹) could interfere the bacterial arrangement of KPN biofilm and extracellular matrix. GML and its CMM & WMM could inhibit the formation of KPN biofilm, CMM & WMM in GML showed synergism and complementation in inhibit KPN biofilm. Results showed that GML had obvious anti-inflammatory and antipyretic effects and could destruct KPN mature biofilm. WMM and CMM showed obvious synergistic effect against inflammation and inhibition of KPN biofilm formation and reduction of number of viable cells but no same effects against fever.