Phenotypic and molecular evaluation of biofilm formation in Klebsiella pneumoniae carbapenemase (KPC) isolates obtained from a hospital of Pelotas, RS, Brazil

Introduction. A significant cause of mortality in the intensive care unit (ICU) is multidrug-resistant (MDR) Gram-negative bacteria, such as Klebsiella pneumoniae carbapenemase (KPC). Biofilm production is a key factor in KPC colonization and persistence in the host, making the treatment difficult. Gap Statement. The aim of this study was to evaluate the antibiotic resistance, molecular and phenotypic biofilm profiles of 12 KPC isolates associated with nosocomial infection in a hospital in Pelotas, Rio Grande do Sul, Brazil. Methodology. Clinical isolates were obtained from different sources, identified and characterized by antibiotic resistance and carbapenemase synthesis following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Polymerase chain reaction (PCR) was used to evaluate the presence of carbapenemase (blaKPC ) and biofilm formation-associated genes (fimA, fimH, rmpA, ecpA, mrkD and wabG). Additionally, phenotypic evaluation of in vitro biofilm formation capacity was evaluated by Congo red agar (CRA) assay and the crystal violet staining method. Results. The 12 isolates evaluated in this study presented the blaKPC gene and were positive for synthesizing carbapenemases in vitro. In the carbapenem class, 83.3 % isolates were resistant and 16.7 % intermediately resistant to imipenem and meropenem. Molecular analyses found that the fimA and wabG genes were detected in 75 % of isolates, while fimH and ecpA were detected in 42 % and mrkD were detected in 8.3 % (1). The CRA assay demonstrated that all isolates were slime producers and 91.7 % (11) of isolates were classified as strong and 8.3 % (1) as moderate biofilm producers by the crystal violet staining method. The optical density (OD540nm) for strong biofilm formers ranged from 0.80±0.05 to 2.47±0.28 and was 0.55±0.12 for moderate biofilm formers. Conclusion. Our study revealed a high level of antibiotic resistance and biofilm formation in KPC isolates obtained from a hospital in Pelotas, RS, Brazil.

Attenuation of Quorum Sensing Controlled Virulence Factors and Biofilm Formation by Edible Fruit Extract of Coccinia indica against Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is a Gram-negative opportunistic human pathogen that mainly infects immunocompromised individuals and patients with urinary tract infection and chronic infections of the respiratory pathways, including cystic fibrosis. Many quorum sensing (QS) controlled components such as bio surfactants and swarming motilities play an important role in the establishment of biofilms. Targeting these factors through anti-QS strategies prevent biofilm formation and treating infections. Coccinia indica commonly called little gourd is used to treat diabetes, wound, burn infections and has antioxidant, antibacterial and antitussive properties. Methods: The methanolic fruit extract of C. indica was prepared and screened for anti-QS and anti-biofilm formation activity. Pyocyanin inhibition, rhamnolipid, crystal violet staining assay tests was performed and the extract was observed under fluorescent microscope. Results: The results obtained are as follows - the fruit extract inhibits the pyocyanin at 58.13% and 42.27% at 0.5 mg/ml and 1.0 mg/ml, biofilm at 69.86% and 49.06% at 0.5 mg/ml and 1.0 mg/ml, inhibits rhamnolipid assay and under fluorescent microscope it is seen scattered whereas control produce biofilm matrix like appearance. Conclusion: Since less study has been made on the quorum sensing and biofilm activity of C.indica our study aimed to fulfil it and it was found that it exhibits good biofilm formation and thus can be used for treating infections.

Synergy between Indoloquinolines and Ciprofloxacin: An Antibiofilm Strategy against Pseudomonas aeruginosa

Antibiotic treatments can participate in the formation of bacterial biofilm in case of under dosage. The interest of indoloquinoline scaffold for drug discovery incited us to study the preparation of new indolo [2,3-b]quinoline derivatives by a domino radical process. We tested the effect of two different “indoloquinoline” molecules (Indol-1 and Indol-2) without antimicrobial activity, in addition to ciprofloxacin, on biofilm formation thanks to crystal violet staining and enumeration of adhered bacteria. This association of ciprofloxacin and Indol-1 or Indol-2 attenuated the formation of biofilm up to almost 80% compared to ciprofloxacin alone, or even prevented the presence of adhered bacteria. In conclusion, these data prove that the association of non-antimicrobial molecules with an antibiotic can be a solution to fight against biofilm and antibiotic resistance emergence.

In vitro Streptococcus mutans adhesion and biofilm formation on different esthetic orthodontic archwires

ABSTRACT Objectives To evaluate the ability of different esthetic archwires to retain oral biofilms in vitro. Materials and Methods Seven different brands of coated orthodontic archwires were tested: two epoxy coated, two polytetrafluoroethylene coated, two rhodium coated, and one silver plus polymer coated. Conventional uncoated metallic archwires were used as controls. Streptococus mutans adherence to archwires was quantified by colony count following 24 hours of biolfilm growth, and total wire-associated biofilm was measured using a crystal violet staining assay. For both tests, two conditions were used: 0% sucrose and 3% sucrose. For statistical analysis, P < .05 was considered as statistically significant. Results For S. mutans colony forming units per biofilm, there were no statistically significant differences among the various archwires (P = .795 for 0% sucrose; P = .905 for 3% sucrose). Regarding total biofilm formed on archwires in the 3% sucrose condition, there were statistically significant differences in crystal violet staining only for the comparison between Niti Micro Dental White and Copper Ni-Ti wires (P < .05). Conclusions The clinical use of esthetic-coated orthodontic wires may be considered to have similar risks as uncoated archwires for biofilm retention.

Antibiotic resistance, biofilm formation, and biofilm-associated genes among Stenotrophomonas maltophilia clinical isolates

Objective The purpose of the present study was to investigate the antimicrobial susceptibility pattern, biofilm production, and the presence of biofilm genes among the S. maltophilia clinical isolates. A total of 85 clinical isolates of S. maltophilia were collected from patients referred to several hospitals. Susceptibility to antibiotics was investigated by disc diffusion method according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). By the crystal violet staining method, the capability of biofilm formation was examined. The genes associated with biofilm production were investigated by the PCR-sequencing techniques. Results All isolates were resistant to doripenem, imipenem, and meropenem. Minocycline, trimethoprim/sulfamethoxazole and levofloxacin exhibited the highest susceptibility of 100%, 97.65%, and 95.29%, respectively. The results of crystal violet staining assay showed that all isolates (100%) form biofilm. Moreover, 24 (28.23%), 32 (37.65%), and 29 (34.12%) of isolates were categorized as weak, moderate, and strong biofilm producers, respectively. Biofilm genes including rpfF, spgM and rmlA had an overall prevalence of 89.41% (76/85), 100% (85/85) and 84.71% (72/85), respectively. Rational prescribing of antibiotics and implementation of infection control protocols are necessary to prevent further infection and development of antimicrobial resistance. Combination strategies based on the appropriate antibiotics along with anti-biofilm agents can also be selected to eliminate biofilm-associated infections.

Antibiofilm effect of Theobroma cacao (cacao pod) extract on Aggregatibacter actinomycetemcomitans biofilm in vitro

Successful of periodontal treatment is to eradicate biofilm of bacteria. Aggregatibacter actinomycetemcomitans is a Gram-negative bacterium that have been suggested to be the main causes of periodontal disease. Theobroma cacao (cacao pod) is a medicinal plant that has a broad range of pharmacological effects. The aim of this study was to assess the antibiofilm effect of cacao pod extract against A. actinomycetemcomitans biofilm in vitro. A. actinomycetemcomitans were cultured in Brain Heart Infusion broth. Crystal-violet staining in biofilm assays were used to evaluate the cacao pod extract effect on A. actinomycetemcomitans ATCC 33384 biofilms and 0.2% chlorhexidine-gluconate was used as a positive control. After 24 hours of incubation, the optical density of each well in microtiter plates was measured. The results showed that the biofilm density after incubation with the cacao pod extract was significantly decreased in all concentrations and all incubation times (p<0.05). The most effective concentration for inhibiting biofilm A. actinomycetemcomitans was 100% cacao pod extract and 3 hrs of incubation time (p<0.05) with a 98.9% reduction of biofilm compared to negative control. Cacao pod extract is effective in inhibiting the growth of A. actinomycetemcomitans biofilm.

Evaluation of pathogenesis and biofilm formation ability of Yersinia pestis after 40-day exposure to simulated microgravity

Background: With the increase of manned space missions and the rise of space microbiology, the research of microbes grown under microgravity environment attracts more attentions. The research scope in space microbiology has been extended beyond pathogens directly related to spaceflight Y. pestis, the causative agent of plague, is also of interest to researchers. Results: After Y. pestis strain 201 cultivated for 40 consecutive passages in either simulated microgravity and normal gravity (NG) conditions, the cultures were used to observe the main phenotypic features of Y. pestis. By using crystal violet staining assays, increased biofilm amount was detected in Y. pestis grown under SMG condition. Besides that, the damage degrees of Hela cell caused by SMG-grown Y. pestis were found diminished in relative to those NG condition. Consistent with this observation, death course was delayed in mice infected with SMG-grown Y. pestis, suggesting that microgravity condition could contribute the attenuated virulence. RNA-seq-based transcriptomics analysis showed a total of 219 genes were differentially regulated, of which 92 upregulated and 127 downregulated. We found dozens of virulence-associated genes were downregulated, which partially explained the reduced virulence of Y. pestis under SMG condition. Our study demonstrated that long-term exposure to simulated microgravity influence the pathogenesis and biofilm formation ability of Y. pestis in a different way, which provides a novel avenue to study the mechanism of physiology and virulence in this pathogen.Conclusions: Microgravity enhanced the ability of biofilm formation of Y. pestis. The virulence and cytotoxicity of Y. pestis were reduced under the microgravity environment. The expressions of many virulence-associated genes of Y. pestis were differentially regulated in response to the stimulated microgravity.

Biofilm-Formation-Related Genes csgD and bcsA Promote the Vertical Transmission of Salmonella Enteritidis in Chicken

The contamination of Salmonella Enteritidis in eggs and chicken meat via vertical transmission has become a worldwide public health concern. Biofilm formation by S. Enteritidis further enhances its antibacterial resistance. However, whether genes related to biofilm formation affect the level of vertical transmission is still unclear. Here, S. Enteritidis mutants ΔcsgD, ΔcsgA, ΔbcsA, and ΔadrA were constructed from wild type strain C50041 (WT), and their biofilm-forming ability was determined by Crystal violet staining assay. Then the median lethal dose (LD50) assay was performed to determine the effects of the selected genes on virulence. The bacterial load in eggs produced by infected laying hens via the intraperitoneal pathway or crop gavage was determined for evaluation of the vertical transmission. Crystal violet staining assay revealed that S. Enteritidis mutants ΔcsgD, ΔcsgA, and ΔbcsA, but not ΔadrA, impaired biofilm formation compared with WT strain. Furthermore, the LD50 in SPF chickens showed that both the ΔcsgD and ΔbcsA mutants were less virulent compared with WT strain. Among the intraperitoneally infected laying hens, the WT strain-infected group had the highest percentage of bacteria-positive eggs (24.7%), followed by the ΔadrA group (16%), ΔcsgA group (9.9%), ΔbcsA group (4.5%), and ΔcsgD group (2.1%). Similarly, among the crop gavage chickens, the WT strain group also had the highest infection percentage in eggs (10.4%), followed by the ΔcsgA group (8.5%), ΔadrA group (7.5%), ΔbcsA group (1.9%), and ΔcsgD group (1.0%). Our results indicate that the genes csgD and bcsA help vertical transmission of S. Enteritidis in chickens.