Features of antifungal therapy during long-lasting infectious process: a clinical case of fungal keratitis and profile of antifungal sensitivity based on assessing biofilm formation

Among infectious diseases, opportunistic mycoses hold a special place. There has been accumulating a lot of evidence regarding the clinical and epidemiological aspects of infection caused by Fusarium spp., which global incidence rate among microbial keratitis ranges from 2 to 40% depending on the geographical location of the country. Colonizing mucous membranes, fungi can exist not only in the form of plankton, but form biofilms after surface attachment, which leads to elevated resistance to multiple antifungal agents. Here we describe a clinical case of fungal keratitis due to Fusarium solani by determining profile of the antifungal sensitivity for isolated fungal strains, by taking into account their potential for biofilm formation. We used an F. solani culture isolated from the patient as well as F. solani test culture obtained from the Russian National Collection of Microorganisms. While determining the sensitivity of fungal planktonic cultures to antifungal agents from the azole group (fluconazole, voriconazole), amphotericin B and terbinafine, it was revealed that antimycotics amphotericin B and voriconazole exerted a marked antifungal activity against clinical isolate, whereas the plankton F. solani test culture was more sensitive to all groups of antifungal agents. Due to a long-lasting progressive course of the infectious process and the high biofilm-forming ability of the clinical strain F. solani, the activity of antifungal agents on biofilm cells was modeled and examined in vitro. It was shown that regarding to the fungal biofilms, value of the minimally inhibitory concentration exceeded those for planktonic cultures by 100-fold. The mechanisms of action for antifungal agents on vital parameters of fungal cell structures were analyzed by using confocal laser scanning microscopy after staining samples with propidium iodide and acridine orange for 15 min to detect changes between intact and damaged cell surface. It was found that within the biofilm fungal cells preserved viability even after exposure to high concentrations of antifungals. In addition, despite the fungicidal drug activity at substantial concentrations acting on the biofilm cell membrane, the cell nuclei remained viable. Owing to the presence ot the mechanism of resistance in mycelial fungi shown in the study, it is necessary to take into account and investigate characteristics of biofilms in terms of drug sensitivity that will allow to optimize a choice of antimicrobial therapy.

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Antibiofilm activity of antifungal drugs, including the novel drug olorofim, against Lomentospora prolificans

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa157 ◽

2020 ◽

Cited By ~ 1

Author(s):

Lisa Kirchhoff ◽

Silke Dittmer ◽

Ann-Kathrin Weisner ◽

Jan Buer ◽

Peter-Michael Rath ◽

...

Keyword(s):

Amphotericin B ◽

Biofilm Formation ◽

Laser Scanning ◽

Antifungal Agents ◽

Fungal Infections ◽

Pathogenic Fungus ◽

Antifungal Drugs ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Antibiofilm Activity

Abstract Objectives Patients with immunodeficiency or cystic fibrosis frequently suffer from respiratory fungal infections. In particular, biofilm-associated fungi cause refractory infection manifestations, linked to increased resistance to anti-infective agents. One emerging filamentous fungus is Lomentospora prolificans. Here, the biofilm-formation capabilities of L. prolificans isolates were investigated and the susceptibility of biofilms to various antifungal agents was analysed. Methods Biofilm formation of L. prolificans (n = 11) was estimated by crystal violet stain and antibiofilm activity was additionally determined via detection of metabolically active biofilm using an XTT assay. Amphotericin B, micafungin, voriconazole and olorofim were compared with regard to their antibiofilm effects when added prior to adhesion, after adhesion and on mature and preformed fungal biofilms. Imaging via confocal laser scanning microscopy was carried out to demonstrate the effect of drug treatment on the fungal biofilm. Results Antibiofilm activities of the tested antifungal agents were shown to be most effective on adherent cells whilst mature biofilm was the most resistant. The most promising antibiofilm effects were detected with voriconazole and olorofim. Olorofim showed an average minimum biofilm eradication concentration (MBEC) of 0.06 mg/L, when added prior to and after adhesion. The MBECs of voriconazole were ≤4 mg/L. On mature biofilm the MBECs of olorofim and voriconazole were higher than the previously determined MICs against planktonic cultures. In contrast, amphotericin B and especially micafungin did not exhibit sufficient antibiofilm activity against L. prolificans. Conclusions To our knowledge, this is the first study demonstrating the antibiofilm potential of olorofim against the human pathogenic fungus L. prolificans.

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In Vitro Activity of Chlorhexidine Compared with Seven Antifungal Agents against 98 Fusarium Isolates Recovered from Fungal Keratitis Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02669-18 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 3

Author(s):

Claudy Oliveira dos Santos ◽

Eva Kolwijck ◽

Henrich A. van der Lee ◽

Marlou C. Tehupeiory-Kooreman ◽

Abdullah M. S. Al-Hatmi ◽

...

Keyword(s):

Amphotericin B ◽

Antifungal Agents ◽

Culture Collection ◽

Fungal Keratitis ◽

Molecular Techniques ◽

Temperate Climate ◽

Eye Drops ◽

Fungal Culture ◽

Content Type

ABSTRACT Fungal keratitis is a common but severe eye infection in tropical and subtropical areas of the world. In regions with a temperate climate, the frequency of infection is rising in patients with contact lenses and following trauma. Early and adequate therapy is important to prevent disease progression and loss of vision. The management of Fusarium keratitis is complex, and the optimal treatment is not well defined. We investigated the in vitro activity of chlorhexidine and seven antifungal agents against a well-characterized collection of Fusarium isolates recovered from patients with Fusarium keratitis. The fungus culture collection of the Center of Expertise in Mycology Radboudumc/CWZ was searched for Fusarium isolates that were cultured from cornea scrapings, ocular biopsy specimens, eye swabs, and contact lens fluid containers from patients with suspected keratitis. The Fusarium isolates that were cultured from patients with confirmed keratitis were all identified using conventional and molecular techniques. Antifungal susceptibility testing was performed according to the EUCAST broth microdilution reference method. The antifungal agents tested included amphotericin B, voriconazole, posaconazole, miconazole, natamycin, 5-fluorocytosine, and caspofungin. In addition, the activity of chlorhexidine was determined. The fungal culture collection contained 98 Fusarium isolates of confirmed fungal keratitis cases from 83 Dutch patients and 15 Tanzanian patients. The isolates were collected between 2007 and 2017. Fusarium oxysporum (n = 24, 24.5%) was the most frequently isolated species followed by Fusarium solani sensu stricto (n = 18, 18.4%) and Fusarium petroliphilum (n = 11, 11.2%). Amphotericin B showed the most favorable in vitro inhibition of Fusarium species followed by natamycin, voriconazole, and chlorhexidine, while 5-fluorocytosine, posaconazole, miconazole, and caspofungin showed no relevant inhibiting effect. However, chlorhexidine showed fungicidal activity against 90% of F. oxysporum strains and 100% of the F. solani strains. Our study supports the clinical efficacy of chlorhexidine and therefore warrants its further clinical evaluation for primary therapy of fungal keratitis, particularly in low and middle income countries where fungal keratitis is much more frequent and, currently, antifungal eye drops are often unavailable.

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Effect of antifungals on itraconazole resistant Candida glabrata

Open Life Sciences ◽

10.2478/s11535-010-0021-5 ◽

2010 ◽

Vol 5 (3) ◽

pp. 318-323 ◽

Cited By ~ 1

Author(s):

Soňa Kucharíková ◽

Patrick Dijck ◽

Magdaléna Lisalová ◽

Helena Bujdáková

Keyword(s):

Amphotericin B ◽

Biofilm Formation ◽

Candida Glabrata ◽

Antifungal Agents ◽

Inhibitory Concentration ◽

Clinical Isolates ◽

Azole Resistance ◽

Low Concentrations ◽

Reduction Assay ◽

Very High

AbstractIn the last decade, infections caused by Candida glabrata have become more serious, particularly due to its decreased susceptibility to azole derivatives and its ability to form biofilm. Here we studied the resistance profile of 42 C. glabrata clinical isolates to different azoles, amphotericin B and echinocandins. This work was also focused on the ability to form biofilm which plays a role in the development of antifungal resistance. The minimal inhibitory concentration testing to antifungal agents was performed according to the CLSI (Clinical and Laboratory Standards Institute) M27-A3 protocol. Quantification of biofilm was done by XTT reduction assay. All C. glabrata clinical isolates were resistant to itraconazole and sixteen also showed resistance to fluconazole. All isolates remained susceptible to voriconazole. Amphotericin B was efficient in a concentration range of 0.125–1 mg/L. The most effective antifungal agents were micafungin and caspofungin with the MIC100 values of ≤0.0313–0.125 mg/L. Low concentrations of these agents reduced biofilm formation as well. Our results show that resistance of different C. glabrata strains is azole specific and therefore a single azole resistance cannot be assumed to indicate general azole resistance. Echinocandins proved to have very high efficacy against clinical C. glabrata strains including those with ability to form biofilm.

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Combination of antifungal drugs and protease inhibitors prevent Candida albicans biofilm formation and disrupt mature biofilms

10.1101/2020.02.11.945030 ◽

2020 ◽

Author(s):

Matthew B. Lohse ◽

Megha Gulati ◽

Charles S. Craik ◽

Alexander D. Johnson ◽

Clarissa J. Nobile

Keyword(s):

Candida Albicans ◽

Protease Inhibitors ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Antifungal Drugs ◽

Aspartyl Protease ◽

Abiotic Surfaces ◽

Drug Concentrations ◽

Aspartyl Protease Inhibitors

AbstractBiofilms formed by the fungal pathogen Candida albicans are resistant to many of the antifungal agents commonly used in the clinic. Previous reports suggest that protease inhibitors, specifically inhibitors of aspartyl proteases, could be effective antibiofilm agents. We screened three protease inhibitor libraries, containing a total of 80 compounds for the abilities to prevent C. albicans biofilm formation and to disrupt mature biofilms. The compounds were screened individually and in the presence of subinhibitory concentrations of the most commonly prescribed antifungal agents for Candida infections: fluconazole, amphotericin B, or caspofungin. Although few of the compounds affected biofilms on their own, seven aspartyl protease inhibitors inhibited biofilm formation when combined with amphotericin B or caspofungin. Furthermore, nine aspartyl protease inhibitors disrupted mature biofilms when combined with caspofungin. These results suggest that the combination of standard antifungal agents together with specific protease inhibitors may be useful in the prevention and treatment of C. albicans biofilm infections.ImportanceCandida albicans is one of the most common pathogens of humans. C. albicans forms biofilms, structured communities of cells several hundred microns thick, on both biotic and abiotic surfaces. These biofilms are typically resistant to antifungal drugs at the concentrations that are normally effective against free-floating cells, thus requiring treatment with higher drug concentrations that often have significant side effects. Here, we show that certain combinations of existing antifungal agents with protease inhibitors, including several drugs already commonly used to treat HIV patients, are effective at inhibiting biofilm formation by C. albicans and/or at disrupting mature C. albicans biofilms.

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In vitroactivities of antifungals alone and in combination with tigecycline againstCandida albicansbiofilms

PeerJ ◽

10.7717/peerj.5263 ◽

2018 ◽

Vol 6 ◽

pp. e5263 ◽

Cited By ~ 6

Author(s):

Mayram Hacioglu ◽

Ayse Seher Birteksoz Tan ◽

Sibel Dosler ◽

Nese Inan ◽

Gulten Otuk

Keyword(s):

Candida Albicans ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Synergistic Effects ◽

Planktonic Cells ◽

Effective Agent ◽

Highly Active ◽

Highly Effective ◽

Anti Fungal

BackgroundCandidamay form biofilms, which are thought to underlie the most recalcitrant infections.MethodsIn this study, activities of antifungal agents alone and in combination with tigecycline against planktonic cells and mature and developing biofilms ofCandida albicansisolates were evaluated.ResultsAmphotericin B and echinocandins were found to be the most effective agents against mature biofilms, whereas the least effective agent was fluconazole. Furthermore, the most effective anti-fungal monotherapies against biofilm formation were amphotericin B and anidulafungin, and the least effective monotherapy was itraconazole. The combination of tigecycline and amphotericin B yielded synergistic effects, whereas combinations containing itraconazole yielded antagonist effects against planktonic cells. The combination of tigecycline and caspofungin exhibited maximum efficacy against mature biofilms, whereas combinations containing itraconazole exhibited minimal effects. Combinations of tigecycline with amphotericin B or anidulafungin were highly effective againstC. albicansbiofilm formation.DiscussionIn summary, tigecycline was highly active againstC. albicansparticularly when combined with amphotericin B and echinocandins.

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Susceptibility of Cryptococcus neoformans Biofilms to Antifungal Agents In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.3.1021-1033.2006 ◽

2006 ◽

Vol 50 (3) ◽

pp. 1021-1033 ◽

Cited By ~ 109

Author(s):

Luis R. Martinez ◽

Arturo Casadevall

Keyword(s):

Confocal Microscopy ◽

Cryptococcus Neoformans ◽

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Capsular Polysaccharide ◽

Antifungal Drugs ◽

Enzyme Linked Immunosorbent Assay ◽

Sequence Of Events ◽

Biofilm Development

ABSTRACT Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.

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Effect of antifungal agents, lysozyme and human antimicrobial peptide LL-37 on clinical Candida isolates with high biofilm production

Journal of Medical Microbiology ◽

10.1099/jmm.0.001283 ◽

2020 ◽

Author(s):

Yi-Chun Chen ◽

Fang-Ju Chen ◽

Chen-Hsiang Lee

Keyword(s):

Amphotericin B ◽

Biofilm Formation ◽

Antifungal Agents ◽

Synergistic Effects ◽

Antifungal Drugs ◽

Planktonic Cells ◽

Xtt Assay ◽

Biofilm Production ◽

Visual Reading

Introduction. Candida species can form biofilms on tissues and medical devices, making them less susceptible to antifungal agents. Hypothesis/Gap Statement. Antifungal combination may be an effective strategy to fight against Candida biofilm. Aim. In this study, we investigated the in vitro activity of fluconazole, caspofungin and amphotericin B, alone and in combination, against 17 clinical Candida tropicalis and 6 Candida parapsilosis isolates with high biofilm formation. We also tested LL-37 and lysozyme for anti-biofilm activity against a selected C. tropicalis isolate. Methodology. Candida biofilms were prepared using the 96-well plate-based method. The minimum biofilm eradication concentrations were determined for single and combined antifungal drugs. The activity of LL-37 and lysozyme was determined by visual reading for planktonic cells and using the XTT assay for biofilms. Results. Under biofilm conditions, fluconazole plus caspofungin showed synergistic effects against 60.9% (14 of 23) of the tested isolates, including 70.6% of C. tropicalis [fractional inhibitory concentration index (FICI), 0.26–1.03] and 33.3% of C. parapsilosis (FICI, 0.04–2.03) isolates. Using this combination, no antagonism was observed. Amphotericin B plus caspofungin showed no effects against 78.3% (18 of 23) of the tested isolates. Amphotericin B plus fluconazole showed no effects against 65.2% (15 of 23) of the tested isolates and may have led to antagonism against 2 C. tropicalis and 2 C. parapsilosis isolates. LL-37 and lysozyme had no effect on biofilms of the selected C. tropicalis isolate. Conclusions. We found that fluconazole plus caspofungin led to a synergistic effect against C. tropicalis and C. parapsilosis biofilms. The efficacy of the antifungal combination therapies of the proposed schemes against biofilm-associated Candida infections requires careful and constant evaluation.

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Microscopic Evaluation, Molecular Identification, Antifungal Susceptibility, and Clinical Outcomes inFusarium,Aspergillusand, Dematiaceous Keratitis

BioMed Research International ◽

10.1155/2013/605308 ◽

2013 ◽

Vol 2013 ◽

pp. 1-10 ◽

Cited By ~ 19

Author(s):

Devarshi U. Gajjar ◽

Anuradha K. Pal ◽

Bharat K. Ghodadra ◽

Abhay R. Vasavada

Keyword(s):

Clinical Outcomes ◽

Amphotericin B ◽

Molecular Identification ◽

Antifungal Agents ◽

Antifungal Susceptibility ◽

Its Region ◽

Fungal Species ◽

Fungal Keratitis ◽

Accurate Identification ◽

Microscopic Evaluation

Purpose.Fusarium,Aspergillus, and Dematiaceous are the most common fungal species causing keratitis in tropical countries. Herein we report a prospective study on fungal keratitis caused by these three fungal species.Methodology. A prospective investigation was undertaken to evaluate eyes with presumed fungal keratitis. All the fungal isolates (n=73) obtained from keratitis infections were identified using morphological and microscopic characters. Molecular identification using sequencing of the ITS region and antifungal susceptibility tests using microdilution method were done. The final clinical outcome was evaluated in terms of the time taken for resolution of keratitis and the final visual outcome. The results were analyzed after segregating the cases into three groups, namely,Fusarium,Aspergillus, and Dematiaceous keratitis.Results. Diagnosis of fungal keratitis was established in 73 (35.9%) cases out of 208 cases. The spectra of fungi isolated wereFusariumspp. (26.6%),Aspergillusspp. (21.6%), and Dematiaceous fungi (11.6%). The sequence of the ITS region could identify theFusariumandAspergillusspecies at the species complex level, and the Dematiaceous isolates were accurately identified. Using antifungal agents such as fluconazole, natamycin, amphotericin B, and itraconazole, the minimum inhibitory concentrations (MICs) forFusariumspp. were >32 μg/mL, 4–8 μg/mL, 0.5–1 μg/mL, and >32 μg/mL, respectively. Antifungal susceptibility data showed thatCurvulariaspp. was highly resistant to all the antifungal agents. Overall, natamycin and amphotericin B were found to be the most effective antifungal agents. The comparative clinical outcomes in all cases showed that the healing response in terms of visual acuity of the Dematiaceous group was significantly good when compared with theFusariumandAspergillusgroups (P<0.05). The time required for healing in theFusariumgroup was statistically significantly less when compared with theAspergillusand Dematiaceous groups.Conclusion. This study demonstrates important differences in microscopic features of scraping material and antifungal susceptibility between the three groups. Early and accurate identification coupled with the MIC data, and thereby appropriate treatment is crucial for complete recovery.

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Antifungal sensitivity profile of Fusarium spp. resulting keratitits

Archives of Infectious Diseases & Therapy ◽

10.33140/aidt/01/01/00005 ◽

2017 ◽

Vol 1 (1) ◽

Keyword(s):

Amphotericin B ◽

Antifungal Agents ◽

Antifungal Susceptibility ◽

Morphological Characteristics ◽

Fungal Keratitis ◽

Morphological Identification ◽

Fusarium Spp ◽

Sensitivity Testing ◽

Resistant Strains ◽

Emergence Of Resistance

Fungal keratitis is an important cause of visual impairment and blindness. Genus Fusarium is a leading cause for fungal keratitis and it has higher degree of resistance to antifungal agents. Our objectives were to identify Fusarium spp. isolated from corneal specimens (received at Dept. of Mycology - MRI from 2013-2016) up to species level and to determine antifungal susceptibility pattern of them. All Fusarium isolates (51) obtained from specimens of patients with keratitis were included in the study. Speciation was done using morphological characteristics of fungi. Antifungal sensitivity testing was done according to CLSI M 51- A guideline, against amphotericin B (10 µg), itraconazole (10 µg). and voriconazole (1 µg). Majority of the isolates were F. solani complex (n=24). Three isolates were difficult to speciate morphologically. Significant number of Fusarium isolates had inhibitory zone diameters (IZD) less than tentative zone diameter epidemiological cut off values (TZD ECVs) for both itraconazole and amphotericin B, indicating emergence of resistant strains against these drugs. Forty five isolates (97.82%) had IZD more than corresponding TZD ECV for voriconazole. All F. solani complexes had IZD less than TZD ECVs for itraconazole. Morphological identification cannot be used as the only method for speciation of Fusarium isolates. Antifungal sensitivity testing should be done for Fusarium isolates from keratitis patients as emergence of resistance strains is not uncommon against commonly used antifungal agents.

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Characterization of biofilm production in different strains of Acinetobacter baumannii and the effects of chemical compounds on biofilm formation

PeerJ ◽

10.7717/peerj.9020 ◽

2020 ◽

Vol 8 ◽

pp. e9020

Author(s):

Ming-Feng Lin ◽

Yun-You Lin ◽

Chung-Yu Lan

Keyword(s):

Acinetobacter Baumannii ◽

Nosocomial Infections ◽

Biofilm Formation ◽

Chemical Compounds ◽

Laser Scanning Microscopy ◽

Hospital Environment ◽

Clinical Strain ◽

Wild Type ◽

Biofilm Production ◽

Different Strains

Acinetobacter baumannii, an important emerging pathogen of nosocomial infections, is known for its ability to form biofilms. Biofilm formation increases the survival rate of A. baumannii on dry surfaces and may contribute to its persistence in the hospital environment, which increases the probability of nosocomial infections and outbreaks. This study was undertaken to characterize the biofilm production of different strains of A. baumannii and the effects of chemical compounds, especially antibiotics, on biofilm formation. In this study, no statistically significant relationship was observed between the ability to form a biofilm and the antimicrobial susceptibility of the A. baumannii clinical isolates. Biofilm formation caused by A. baumannii ATCC 17978 after gene knockout of two-component regulatory system gene baeR, efflux pump genes emrA/emrB and outer membrane coding gene ompA revealed that all mutant strains had less biofilm formation than the wild-type strain, which was further supported by the images from scanning electron microscopy and confocal laser scanning microscopy. The addition of amikacin, colistin, LL-37 or tannic acid decreased the biofilm formation ability of A. baumannii. In contrast, the addition of lower subinhibitory concentration tigecycline increased the biofilm formation ability of A. baumannii. Minimum biofilm eradication concentrations of amikacin, imipenem, colistin, and tigecycline were increased obviously for both wild type and multidrug resistant clinical strain A. baumannii VGH2. In conclusion, the biofilm formation ability of A. baumannii varied in different strains, involved many genes and could be influenced by many chemical compounds.

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