Congo Red Method Research Articles

Identification and Growth Characteristics of Cellulase Producing Bacteria

This study was envisaged to from fresh cow dung using the Congo red staining method, and screened by measuring their endoglucanase activity (CMC enzyme activity), exoglucanase activity (Cex enzyme activity), and filter paper enzyme activity (FPA enzyme activity). Subsequently, the molecular, morphological characteristics, and growth characteristics were studied. Three strains showed clear transparent circles on the Congo red cellulose medium. LY3 exhibited strong enzyme production ability, with relatively higher enzyme activity than the other two strains. LY3 was found to be a dominant strain that required a temperature of 40℃and pH 7.0 for optimal growth. Based on the molecular and morphological characteristics, the three strains were preliminarily identified as Bacillus. Bangladesh J. Bot. 53(3): 849-856, 2024 (September) Special

Fibrobacter sp. HC4, a newly isolated strain, demonstrates a high cellulolytic activity as revealed by enzymatic measurements and in vitro assay.

Despite their low quantity and abundance, the cellulolytic bacteria that inhabit the equine large intestine are vital to their host, as they enable the crucial use of forage-based diets. Fibrobacter succinogenes is one of the most important intestinal cellulolytic bacteria. In this study, Fibrobacter sp. HC4, one cellulolytic strain newly isolated from the horse cecum, was characterized for its ability to utilize plant cell wall fibers. Fibrobacter sp. HC4 consumed only cellulose, cellobiose, and glucose and produced succinate and acetate in equal amounts. Among genes coding for CAZymes, 26% of the detected glycoside hydrolases (GHs) were involved in cellulolysis. These cellulases belong to the GH5, GH8, GH9, GH44, GH45, and GH51 families. Both carboxymethyl cellulase and xylanase activities of Fibrobacter sp. HC4 were detected using the Congo red method and were higher than those of F. succinogenes S85, the type strain. The in vitro addition of Fibrobacter sp. HC4 to a fecal microbial ecosystem of horses with large intestinal acidosis significantly enhanced fibrolytic activity as measured by the increase in gas and volatile fatty acids production during the first 48 h. According to this, the pH decreased and the disappearance of dry matter increased at a faster rate with Fibrobacter sp. HC4. Our data suggest a high specialization of the new strain in cellulose degradation. Such a strain could be of interest for future exploitation of its probiotic potential, which needs to be further determined by in vivo studies.IMPORTANCECellulose is the most abundant of plant cell wall fiber and can only be degraded by the large intestine microbiota, resulting in the production of volatile fatty acids that are essential for the host nutrition and health. Consequently, cellulolytic bacteria are of major importance to herbivores. However, these bacteria are challenged by various factors, such as high starch diets, which acidify the ecosystem and reduce their numbers and activity. This can lead to an imbalance in the gut microbiota and digestive problems such as colic, a major cause of mortality in horses. In this work, we characterized a newly isolated cellulolytic strain, Fibrobacter sp. HC4, from the equine intestinal microbiota. Due to its high cellulolytic capacity, reintroduction of this strain into an equine fecal ecosystem stimulates hay fermentation in vitro. Isolating and describing cellulolytic bacteria is a prerequisite for using them as probiotics to restore intestinal balance.

Determine Biofilm Genes in Pseudomonas Aeruginosa Isolated from Clinical and Environmental Samples

Objective: Isolates of Pseudomonas aeruginosa are an extremely adaptable bacterium that causes opportunistic diseases because of its varied metabolic pathways, genes, virulence factors, and considerable antibiotic resistance. Methods: A total of 293 samples were collected from different places: 193 samples (% 65.87) of human samples and 100 samples (% 34.13) of wastewater samples in the period between 3rd September to 15th November 2023). Bacterial isolates were identified according to microscopic, cultural, and genetic characteristics. Antibiotic susceptibility of bacterial isolates was determined against twelve of the selected antibiotics. The biofilm production was done by using phenotypic ways (Congo red agar and Microtiter plate methods) as well as genotypic ways by detection of biofilm genes (algD, pelf, and pslD genes). Results: Hundred-forty eight bacterial isolates were obtained, and sixty of these isolates were identified as Pseudomonas spp. (40.9%), twenty-six isolates of E. coli (17.9%), seventeen isolates of K. pneumoniae (11.3%), and forty-five isolates were beyond to other types of bacteria (30.1%), and out of sixty isolates of Pseudomonas spp., forty-two were identified as P. aeruginosa isolates. P. aeruginosa isolates revealed various resistance levels to antimicrobial agents gradually, ranging from 83.87% to Trimethoprim-sulfamethoxazole (SXT) to 3.22% to Aztreonam (AZT). Biofilm production by using the Congo red method showed that 27 isolates (64.28%) were positive results, while in the microtiter method, all forty-two isolates were positive (100%), the genetic detection showed that the AlgD gene was recognized in thirty-one isolates (73.8%), followed by Pelf and PslD genes in four isolates each (9.5%). Conclusion: The isolation percentage showed a high occurrence of multi-drug resistance biofilm forming Pseudomonas spp. isolates which could be a critical indicator. Methods of biofilm detection showed that the microtiter plate method has accuracy more than the Congo red method; as well AlgD gene was prevalent compared with both other genes Pelf, and PslD.

Antifungal susceptibility and virulence determinants profile of candida species isolated from patients with candidemia

Candida is the most prevalent fungal bloodstream infection (BSI) with a high mortality rate among hospitalized patients. Another concern facing physicians is rising global incidence of drug-resistant Candida. This study aimed to characterize the prevalence, antifungal susceptibility, biofilm formation, and virulence genes (HWP1, ALS1, SAP2) of different Candida spp. isolated from patients with candidemia. 52 isolates of Candida spp. were identified from blood cultures by chromogenic Candida agar and confirmed by the VITEK 2 system. Isolates were tested for antifungal susceptibility by disk diffusion and VITEK 2 system. Biofilm formation and investigated genes were detected by the Congo red method and conventional PCR, respectively. Candida spp. caused 2.3% of detected BSIs, of which 32.7% were caused by Candida albicans (C. albicans) and 67.3% by non-albicans Candida (NAC), with the predominance of C. tropicalis (25%), followed by C. parapsilosis (17.3%), and C. krusei (13.5%). The susceptibility rates to fluconazole, voriconazole, caspofungin, micafungin, amphotericin B, and flucytosine were 64.7%, 76.5%, 100.0%, 100%, 100.0%, and 100.0% in C. albicans, while 53.6%, 71.4%, 91.4%, 91.4%, 94.3%, and 94.3% in NAC, respectively. Biofilm production, HWP1, ALS1, and SAP2 were detected in 70.6%, 82.4%, 76.5%, and 52.9% of C. albicans and 74.3%, 85.7%, 80.0%, and 48.6% of NAC, respectively. There is remarkable shift to NAC BSIs and high azole resistance. Antifungal stewardship and analysis of risk factors associated with this shift are needed.

Antibacterial and Antibiofilm Potential of Bacterial Cellulose Hydrogel Containing Vancomycin against Multidrug-Resistant Staphylococcus aureus and Staphylococcus epidermidis.

The present study aimed to evaluate the in vitro antibacterial and antibiofilm activity of bacterial cellulose hydrogel produced by Zoogloea sp. (HYDROGEL) containing vancomycin (VAN) against bacterial strains that cause wound infections, such as multidrug-resistant (MDR) Staphylococcus aureus and Staphylococcus epidermidis. Initially, HYDROGEL was obtained from sugar cane molasses, and scanning electron microscopy (SEM) was performed to determine morphological characteristics. Then, VAN was incorporated into HYDROGEL (VAN-HYDROGEL). The antibacterial activity of VAN, HYDROGEL, and VAN-HYDROGEL was assessed using the broth microdilution method to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) against methicillin-sensitive S. aureus (MSSA) ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 33591, S. epidermidis INCQS 00016 (ATCC 12228), five clinical isolates of MRSA, and nine clinical isolates of methicillin-resistant S. epidermidis, following the Clinical and Laboratory Standards Institute (CLSI) guidelines. Additionally, the antibacterial activity of VAN, HYDROGEL, and VAN-HYDROGEL was studied using the time-kill assay. Subsequently, the antibiofilm activity of VAN, HYDROGEL, and VAN-HYDROGEL was evaluated using crystal violet and Congo red methods, as well as SEM analysis. VAN and VAN-HYDROGEL showed bacteriostatic and bactericidal activity against MRSA and methicillin-resistant S. epidermidis strains. HYDROGEL did not show any antibacterial activity. Analysis of the time-kill assay indicated that HYDROGEL maintained the antibacterial efficacy of VAN, highlighting its efficiency as a promising carrier. Regarding antibiofilm activity, VAN and HYDROGEL inhibited biofilm formation but did not demonstrate biofilm eradication activity against methicillin-resistant S. aureus and S. epidermidis strains. However, it was observed that the biofilm eradication potential of VAN was enhanced after incorporation into HYDROGEL, a result also proven through images obtained by SEM. From the methods carried out in this study, it was possible to observe that HYDROGEL preserved the antibacterial activity of vancomycin, aside from exhibiting antibiofilm activity and enhancing the antibiofilm effect of VAN. In conclusion, this study demonstrated the potential of HYDROGEL as a candidate and/or vehicle for antibiotics against MDR bacteria that cause wound infections.

Highly efficient nanocellulose adsorbents

Adapting the use of nanocellulose as a sorbent is currently considered an extremely pressing problem, as synthetic analogues, although perform their functions well, are often toxic and incapable of biodegradation. In addition, the production of such synthetic sorbents is quite expensive. Therefore, today the production and modification of nanocellulose in order to increase their absorption capacity is especially important. Furthermore, a number of new applications of nanocellulose have been presented in nanocomposites, Pickering emulsions, wood adhesives, wastewater treatment, and emerging biomedical technologies. The problems and possibilities of new materials based on nanocellulose are being actively discussed. In the course of this work, a composite based on nanocrystalline cellulose from buckwheat, characterized by high sorption properties, environmental safety, and low cost of production, was obtained. Modification of cellulose was carried out with lactic acid. Nine groups of nanocrystalline cellulose samples with different acid soaking times were synthesized, and the optimal time required for the complete grafting of the acid into cellulose was determined. The specific surface area of the composite material was calculated using the Congo red dye adsorption method. It has been found that the specific surface area of the modified composite is not inferior to the specific surface area of many synthetic adsorbents, and even surpasses them in terms of environmental and economic indicators.

WITHDRAWN: Isolation and Functional Study of Efficient Cellulose Degrading Bacteria from Horse Cecum Contents

ObjectivesAnimals that consume and digest roughage for a long time have unique gut microbiota in their gastrointestinal tract. Studying the isolation of beneficial functional bacteria from the gut can provide resources for the development of feed microbial additives. Therefore, the purpose of this study was to isolate and identify bacteria with cellulose degradation ability from the cecal contents of Yili horses, and to provide resources for the development of feed microbial additives. MethodsFirstly, the cecal contents of 18 Yili horses were collected, and cellulose degrading bacteria were isolated and screened using CMC-Na screening medium under aerobic and anaerobic conditions. The strains with strong cellulose degradation ability were identified using Congo red staining and DNS methods. The top 60 strains with enzyme index were selected, and full-length 16S rRNA sequencing technology was used to identify and construct a phylogenetic tree. Select the top 5 strains of bacteria with high enzyme activity based on the measured cellulase activity, and the sheep were treated with mixed bacterial suspension by gavage. The rumen microbiota of sheep was analyzed using 16S rDNA amplicon sequencing technology. ResultsA total of 263 single colonies were isolated, of which 120 single colonies had a hydrolysis circle and colony enzymatic index (EI) of >1.5. Enzyme activity determination screening of the top 60 strains of full-length 16S rRNA sequencing, sequencing results show that the strains are mostly Bacillus spp. Among the 60 strains of bacteria, CE207 has the highest enzyme activity of 14.81 U/mL, and the lowest enzyme activity of CE105 is 1.91 U/mL. The mixed bacteria significantly increased the daily weight gain of sheep (P < 0.001) and feed intake (P < 0.001), and feed conversion rate increased from 7.75% to 10.09%. Sequencing results of sheep rumen microorganisms showed that, compared with the control group, the experimental group contained Muribaculum, Parvibacter, Desulfovibrio, Novosphingobium, Cutibacterium, Parasutterella, Lachnospiraceae_UCG-006, and Allobaculum genera increased significantly in abundance, and Fibrobacter and NK4A214_group genera decreased significantly in abundance, suggesting that a mixture of bacteria can influence the changes in the microflora of the rumen intestinal tract of sheep. ConclusionsThis finding suggests that the cellulose degrading bacteria isolated in the study can effectively promote the growth and development of herbivores such as sheep, and that efficient cellulose degrading bacteria in the animal gut may become a new direction for the development of feed additives in the future.

Observations of amyloid breakdown by proteases over time using scanning acoustic microscopy

Amyloid consists of insoluble beta-fibrillar proteins with stable structures. The Congo red staining method for histologically detecting amyloid is unsuitable for quantitatively assessing amyloid fibers. Scanning acoustic microscopy (SAM) detects the attenuation of sound (AOS) through sections. This study aimed to clarify whether AOS values reflected the amount of amyloid fibril degradation in tissues. Formalin-fixed paraffin-embedded unstained sections of various types of amyloidosis were digested with different endopeptidases. The AOS images after digestion were observed over time via SAM. The corresponding Congo red-stained images were followed to identify the amyloid. The amyloid and nonamyloid portions were statistically examined over time to determine the changes in the AOS values. Most of the amyloid areas showed significantly different AOS values from nonamyloid portions before digestion and significantly decreased after digestion; these findings corresponded with the disappearance and waning of the Congo red staining in the light microscopic images. Some nonamyloid areas with high AOS masked the reduction in AOS in the amyloid areas. The method used in this study may help detect the amyloid quantity and determine the appropriate treatment method for removing amyloid deposits from tissues.

Isolation, Identification and Assessment of Efficient Cellulase Producing Bacteria from the Termite Guts

The present study is concerned with the screening the cellulase-producing bacteria from termite gut, assessed potential cellulase-producing bacteria and partial characterization (optimum parameters) of cellulase from isolated bacteria. The result showed that 15 out of 48 isolated strains was positive for degrading the carboxy methyl cellulose (CMC) in agar by congo-red method. After screening by DNS assay, three selected bacteria exhibited high cellulase activity that were identified as Citrobacter amalonaticus CM 1-3, Bacillus cereus CM 5-1 and Streptococcus salivarius CE 5-1 using 16S rRNA sequence analysis. All bacterial strains utilized CMC and showed the highest cellulase activity. Cellulase characterization of C. amalonaticus CM 1-3 and S. salivarius CE 5-1 was revealed optimum activity at 35°C, pH 7.0 and for 48 h. Bacillus cereus CM 5-1 represented its potential use in industrial processes due to thermostable cellulase production. The crude cellulase of this strain was purified by (NH4)2SO4 precipitation with 1.58 purification fold and 74.38% overall recovery. The optimal temperature and pH for cellulase activity of B. cereus CM 5-1 were at 40°C and pH 7.0. Thus, this study provided additional information about the diversity and partial characteristic cellulase of cellulolytic bacteria from termite gut for future industrial applications.

Biofilm Formation of Locales Clinical Multidrug Resistance Klebsiella pneumoniae Isolated from Different Sources deals with Antimicrobial Resistance

The goal of this study was to detect antibiotic resistance and determine the ability of clinical isolates Klebsiella pneumoniae (K. pneumoniae) to form biofilms and detect the relationship between biofilm formation and antimicrobial resistance that depends on the specimen sources at the localized Baghdad Hospitals. This gram-negative rod bacterium is an opportunistic pathogen that can cause various illnesses in people and animals, such as important respiratory tract infections. For these reasons, our study included 87 isolates of K. pneumoniae from different clinical cases. The number and percentage of obtained isolates according to the sources distributed as specimens: 26(29.9%) urine, 25(28.7%) blood, 8(9.2%) stool, and 4(4.6%) sputum, as well as swabs:11(12.6%) burn, 9(10.3%) vagina and 4(4.6%) wound. The antibiotic susceptibility test results showed colistin, Imipenem, and meropenem were more effective against the isolates. The tissue culture plate and Congo red methods were used to evaluate biofilm formation. Finally, polymerase chain reaction was used to identify two genes linked with biofilm formation: MrkD and FimH. The isolates showed different abilities to produce biofilms based on clinical sources. The result appeared (97.7%) of isolates as biofilm producers from the following: 41(47.13%) strongly, 33 (37.93%) moderately, and 11 (12.64%) weakly. While only two isolates 2 (2.3 %) represented non-biofilm producers. 100 and 91% of the isolates, respectively, had the MrkD and FimH biofilm formation genes, according to molecular analysis. A recent study showed biofilm formation by K. pneumoniae strains isolated from blood specimens could form stronger biofilms. On the other hand, stool specimens formed weaker biofilm compared to them. According to this study, multidrug-resistant (MDR) K. pneumoniae strains' capacity to form biofilms and their antibiotic resistance profile is positively correlated. These could aid in developing therapeutic therapies for infections brought on by K. pneumoniae resistant to carbapenems, considered the "final line of defense" antibiotics. We can infer from this work that K. pneumoniae could be isolated from many sources and was MDR, as well as having the different capacity to build biofilm in various ways, especially in hospital cases of high antibiotic resistance. Keywords: K. pneumoniae, Biofilm formation, MDR, PDR, XDR, Congo red

Bioinspired fabrication of zinc hydroxide-based nanostructure from lignocellulosic biomass Litchi chinensis leaves and its efficacy evaluation on antibacterial, antioxidant, and anticancer activity

Zinc-based nanostructures are known for their numerous potential biomedical applications. In this context, the biosynthesis of nanostructures using plant extracts has become a more sustainable and promising alternative to effectively replace conventional chemical methods while avoiding their toxic impact. In this study, following a low-temperature calcination process, a green synthesis of Zn-hydroxide-based nanostructure has been performed using an aqueous extract derived from the leaves of Litchi chinensis, which is employed as a lignocellulose waste biomass known to possess a variety of phytocompounds. The biogenic preparation of Zn-hydroxide based nanostructures is enabled by bioactive compounds present in the leaf extract, which act as reducing and capping agents. In order to evaluate its physicochemical characteristics, the produced Zn-hydroxide-based nanostructure has been subjected to several characterization techniques. Further, the multifunctional properties of the prepared Zn-hydroxide-based nanostructure have been evaluated for antioxidant, antimicrobial, and anticancer activity. The prepared nanostructure showed antibacterial efficacy against Bacillus subtilis and demonstrated its anti-biofilm activity as evaluated through the Congo red method. In addition, the antioxidant activity of the prepared nanostructure has been found to be dose-dependent, wherein 91.52 % scavenging activity could be recorded at 200 μg/ml, with an IC50 value of 45.22 μg/ml, indicating the prepared nanostructure has a high radical scavenging activity. Besides, the in vitro cytotoxicity investigation against HepG2 cell lines explored that the as-prepared nanostructure exhibited a higher cytotoxic effect and 73.21 % cell inhibition could be noticed at 25.6 μg/ml with an IC50 of 2.58 μg/ml. On the contrary, it was found to be significantly lower in the case of HEK-293 cell lines, wherein ~47.64 % inhibition could be noticed at the same concentration. These findings might be further extended to develop unique biologically derived nanostructures that can be extensively evaluated for various biomedical purposes.

Study on Application and Mechanism of Alkali Sulfadiazine Neodymium as PVC Heat Stabilizer

In this paper, the alkali sulfadiazine neodymium (NdSD) was prepared from sulfadiazine and neodymium nitrate hexahydrate under alkaline conditions, and used as a heat stabilizer on PVC. The structure of NdSD was characterized by elemental analysis and infrared spectrum. The results show that the molecular formula of NdSD is Nd (SD)2·OH·H2O. The thermal degradation process of NdSD at 25-800°C was characterized by thermogravimetric analysis. The results show that the decomposition temperature of NdSD is higher than 200°C, which is suitable for PVC thermal stabilizer. The thermal stabilizer time of PVC by NdSD was characterized by static Congo red method. The results showed that the thermal stabilizer time of PVC increased from 2.7min to 23.2 min after adding NdSD. The discoloration effect of NdSD on PVC was characterized by oven discoloration method. The results indicate that PVC samples added with NdSD show better effect than other heat stabilizers. By means of silver nitrate solution method and Fourier transform infrared spectroscopy, it was proved that sulfadiazine neodymium can absorb and react with HCl, and further proposed a possible mechanism of NdSD stabilization of PVC.

The Mushroom Glucans: Molecules of High Biological and Medicinal Importance.

Carbohydrates, including polysaccharide macromolecules, are the main constituents of the fungal cell wall. Among these, the homo- or heteropolymeric glucan molecules are decisive, as they not only protect fungal cells but also have broad, positive biological effects on the animal and human bodies. In addition to the beneficial nutritional properties of mushrooms (mineral elements, favorable proteins, low fat and energy content, pleasant aroma, and flavor), they have a high glucan content. Folk medicine (especially in the Far East) used medicinal mushrooms based on previous experience. At the end of the 19th century, but mainly since the middle of the 20th century, progressively more scientific information has been published. Glucans from mushrooms are polysaccharides that contain sugar chains, sometimes of only one kind (glucose), sometimes having several monosaccharide units, and they have two (α and β) anomeric forms (isomers). Their molecular weights range from 104 to 105 Da, and rarely 106 Da. X-ray diffraction studies were the first to determine the triple helix configuration of some glucans. It seems that the existence and integrity of the triple helix structure are criteria for their biological effects. Different glucans can be isolated from different mushroom species, and several glucan fractions can be obtained. The biosynthesis of glucans takes place in the cytoplasm, the processes of initiation and then chain extension take place with the help of the glucan synthase enzyme complex (EC 2.4.1.34), and the sugar units are provided by sugar donor UDPG molecules. The two methods used today for glucan determination are the enzymatic and Congo red methods. True comparisons can only be made using the same method. Congo red dye reacts with the tertiary triple helix structure, and the resulting glucan content better reflects the biological value of glucan molecules. The biological effect of β-glucan molecules is proportional to the integrity of the tertiary structure. The glucan contents of the stipe exceed the values of the caps. The glucan levels of individual fungal taxa (including varieties) differ quantitatively and qualitatively. This review presents in more detail the glucans of lentinan (from Lentinula edodes), pleuran (from Pleurotus ostreatus), grifolan (from Grifola frondose), schizophyllan (from Schizophyllum commune), and krestin (from Trametes versicolor), along with their main biological effects.

Detection of Biofilm Formation in Classical and Hypervirulent Klebsiella pneumoniae

Klebsiella pneumoniae has considered as a relevant healthcare-associated pathogen, its risk of infections is increasing in the presence of medical devices. K. pneumoniae is known for its ability to form biofilm on biotic and abiotic surfaces. Biofilm of K. pneumoniae assists in bacterial protection from host immune responses and antibiotics. Hypervirulent Klebsiella pneumoniae (hvKp) emerges as a new pathotype, which first appeared in Asian Pacific Rim but spread globally. Thus, this study aimed to investigate the ability of K. pneumoniae including hvKp and potential hvKp isolates to form biofilm. One hundred isolates of K. pneumoniae were collected from different hospitals in Baghdad city. These isolates were identified by phenotypic characterization on selective agar plates, biochemical tests, VITEK II, and molecular identification. Biofilm formation was tested in these isolates by two methods, congo red and Tissue Culture Plate method. In congo red method, 33% of the isolates were biofilm producer and (63%) can form biofilm by TCP method divided as: 14% strong, 15 moderate, 34% weak, and 37% non-biofilm producer. The hvKp and potential hvKp isolates showed a variable ability to form biofilm as classical K. pneumoniae.

Detection of PgaABCD Proteins in Biofilm Producing Acinetobacter Baumannii Isolates

Acinetobacter baumannii is a common cause of nosocomial infections. This bacterium is able to survive in hostile environments (desiccation, antimicrobial therapies, nutrient unavailability) beside colonization biotic and abiotic surfaces and form biofilm in hospitals and long-term care institutions. Biofilm is a three-dimensional structure of a multicellular complex in which the cells are embedded in an Extracellular Polymeric Substance (EPS) that was produced by the organism itself. Biofilm matrix contains proteins, ions, nucleic acids, and polysaccharide polymers. The main factor that leads to biofilm formation in A. baumannii is the creation of the exopolysaccharide poly-1,6-N-acetylglucosamine (PNAG), which is a virulence factor required for adhesion and aggregation by many Gram-negative bacteria. Many studies indicated that PNAG is an important factor to keep the integrity of A. baumannii biofilms in a more dynamic and stressful environment. A cluster of four genes (pgaA, pgaB, pgaC, and pgaD) are responsible for PNAG, which is encoded by pgaABCD locus. Therefore, the aim of this work was to detect proteins encoded by this locus using SDS-PAGE gel in A. baumannii isolates. Using PCR analysis to detect 16S rRNA concluded that all 24 isolates showed a positive amplification with 242bp. Acinetobacter baumannii clinical isolates showed high resistance percentage to ampicillin-sulbactam (AMS) and ceftazidime (CAZ) with 100% and 91% respectively. Furthermore, the isolates showed 83.3% for cefepime (FEP) and 70.8% for amikacin (AK), while the isolates showed a variable resistance percentage toward other antibiotics. By using Congo red method indicated that 66.7% were positive to produce biofilm and 33.3 were non-forming biofilm. Protein sequences alignment showed 99%, 99%, 100% and 99% identity for PgaA, PgaB, PgaC and PgaD respectively with protein GenBank database.

Hexanic extract of Achyrocline satureioides: antimicrobial activity and in vitro inhibitory effect on mechanisms related to the pathogenicity of Paenibacillus larvae.

Paenibacillus larvae is a spore-forming bacillus, the most important bacterial pathogen of honeybee larvae and the causative agent of American foulbrood (AFB). Control measures are limited and represent a challenge for both beekeepers and researchers. For this reason, many studies focus on the search for alternative treatments based on natural products. The objective of this study was to determine the antimicrobial activity of the hexanic extract (HE) of Achyrocline satureioides on P. larvae and the inhibitory activity on some mechanisms related to pathogenicity. The Minimum Inhibitory Concentration (MIC) of the HE was determined by the broth microdilution technique and the Minimum Bactericidal Concentration (MBC) by the microdrop technique. Swimming and swarming motility was evaluated in plates with 0.3 and 0.5% agar, respectively. Biofilm formation was evaluated and quantified by the Congo red and crystal violet method. The protease activity was evaluated by the qualitative technique on skim milk agar plates. It was determined that the MIC of the HE on four strains of P. larvae ranged between 0.3 and 9.37µg/ml and the MBC between 1.17 and 150µg/ml. On the other hand, sub-inhibitory concentrations of the HE were able to decrease swimming motility, biofilm formation and the proteases production of P. larvae.

Comparative analysis of β-glucosidase activity in non-conventional yeasts.

The objective of this study was to evaluate the β-glucosidase activity in the non-conventional yeasts under cellulose, glucose and sucrose substrates. The participation of the enzyme β-glucosidase and its contribution to the enzymatic degradation of tannins is known. Within the classification of tannins are ellagitannins, molecules of gallic acid and ellagic acid, which are considered as nutraceutical compounds due to the properties that they present and that they can be used in the design of food and new drugs, synthesis of materials with antimicrobial capacity. The extracellular β-glucosidase activity was mainly presented in the Candida and Pichia strains, being the glucose and sucrose media the most capable for inducing the activity that showed maximum values with P. pastoris in glucose (0.1682±0.00 µmol/min mg protein), and C. utilis in cellulose (0.1129±0.1349 µmol/min mg of protein), and sucrose (0.0657±0.0214 µmol/min mg protein). Additionally, I. terricola and P. kluyvery stood out in a qualitative cellulose degradation approach measured by Congo red method (9.60±0.04 mm and 9.20±0.05 mm respectively). These indicate that P. pastoris and C. utilis have potential as β-glucosidase producers, especially when growing under complex carbon sources for biomass conversion, new biofuels production and polyphenol degradation with more manageable bioreactor process.

CELLULASE OF ENDOPHYTIC Bacillus SP. FROM Glycyrrhiza uralensis F. AND ITS APPLICATION FOR EXTRACTION OF GLYCYRRHIZIC ACID

Glycyrrhizic acid is the main component of the medicinal plant Glycyrrhiza uralensis Fisch. It is widely used as a sweetener and an effective active ingredient with multiple physiological functions. Endophytes are microorganisms that coexist with plants and can produce cellulase. This cellulase enzyme can be used to overcome dissolution barriers of plant active ingredients by degrading plant cell wall. In the present study, a cellulase-producing strain with high cellulase activity was isolated from fresh Glycyrrhiza uralensis Fisch, and identified using the Congo red staining method and the DNS method. Glycyrrhizic acid yield was determined by the HPLC method. A highly reactive cellulase-producing strain, with a high extraction capacity of glycyrrhizic acid, was obtained. The strain was named GG-3, and bioinformatic analysis showed that it was a Bacillus sp. Findings obtained after optimization of the enzyme production and glycyrrhizic acid extraction process showed that glycyrrhizic acid yield increased by 32.52% and 31.35% after extraction with GG-3 enzyme, compared with the use of the traditional extraction method and commercial cellulase extraction method, respectively.

Green Synthesis of Zn(OH)2/ZnO-Based Bionanocomposite using Pomegranate Peels and Its Application in the Degradation of Bacterial Biofilm.

The ability and potency of bacterial species to form biofilms, which show antibiotic resistance thereby avoiding antibiotic surfaces, is a major cause of prolonged infections. Various advanced approaches have been employed to prevent or damage bacterial biofilms, formed by a variety of bacterial strains, to help prevent the associated infectious disease. In this context, zinc-based nanostructures have been recognized as a potential antibiotic agent against a broad spectrum of bacterial communities. As a result, a sustainable and green synthesis method was adapted in the present study to synthesize a Zn(OH)2/ZnO-based bionanocomposite, in which aqueous extracts of waste pomegranate peels (Punica granatum) were employed as a natural bioreducing agent to prepare the bionanocomposite at room temperature. Furthermore, FT-IR, XRD, DLS, UV-Visible, PL spectroscopy, FE-SEM, and TEM were used to characterize the green route synthesized a Zn(OH)2/ZnO bionanocomposite. The average crystallite size was determined using the Scherrer relation to be 38 nm, and the DLS results indicated that the Zn(OH)2/ZnO bionanocomposite had a hydrodynamic size of 170 nm. On the other hand, optical properties investigated through UV-Vis and PL spectroscopy explored the energy bandgap between 2.80 and 4.46 eV, corresponding to the three absorption edges, and it covered the blue spectrum when the sample was excited at 370 nm. Furthermore, the impact of this green route synthesized a Zn(OH)2/ZnO bionanocomposite on the biofilm degradation efficiency of the pathogenic bacterial strain Bacillus subtilis PF_1 using the Congored method was investigated. The Congored assay clearly explored the biofilm degradation efficiency in the presence of a 50 mg/mL and 75 mg/mL concentration of the Zn(OH)2/ZnO bionanocomposite against the bacterial strain Bacillus subtilis PF_1 grown for 24 h. This study can be further applied to the preparation of bionanocomposites following a low-cost green synthesis approach, and thus prepared nanostructures can be exploited as advanced antimicrobial agents, which could be of great interest to prevent various infectious diseases.

S1655 Increased Prevalence of Achlorhydria With Age in an Asian Population: Should We Be Screening for Achlorhydria in Patients Aged 50 and over: 22 Years Experience From a Large Hospital

Introduction: Achlorhydria (AC) is a state in which the stomach is unable to produce hydrochloric acid. AC can be categorized as either permanent or temporary based on its causes. It can lead to several conditions including pernicious anemia (PA) which can cause irreversible neurological deficits. Diagnostic techniques of AC can be easily performed during esophagogastroduodenoscopy (EGD) by staining Congo red on the gastric mucosa. Methods: This analysis involved a large single center retrospective study aimed at determining the prevalence of AC in Thailand among EGD patients. Results: A total of 3,597 patients underwent EGD including Congo red staining method at the Vichaiyut Hospital from January 2010 to December 2019; 223 ones were excluded due to concurrent use of proton pump inhibitors. Results revealed that 18 from 3,374 patients (0.53%) had AC. Seven presented with permanent AC (5F, 2M) (median age = 69 years; range 58-92; Table). All 7 patients were found to have high levels of serum gastrin (mean = 2,485 pg/ml; S.D. = 689 pg/ml) and were pathologically confirmed with intestinal metaplasia. All 7 patients with permanent AC were found only when they were over 55 years old. Among 11 patients with temporary AC (5M, 6F: mean age 73.4 years; SD = 13.2 years), all had gastrointestinal Helicobacter pylori bacterial infection after being examined. After successful treatment for H. pylori and re- examination with Congo red, AC was absent among patients with temporary AC. All 11 patients with temporary AC were found only when they were over 45 years old. If counting only patients over 45 years of age, the prevalence of AC was 0.68% (18/2,614). No adverse events arising from Congo red occurred (Figure). Conclusion: Achlorhydria (AC) is relatively rare. Permanent and temporary AC were found only when they were over 55 and 45 years old, respectively. Staining Congo red on gastric mucosa can be safely and routinely incorporated into the EGD procedure for early detection of AC. We recommended a low-cost screening test such as serum vitamin B levels for screening only in patients aged 50 and over.Figure 1.: Congo red changes its color from red to dark blue or black in an acidic environment. Table 1. - Characteristics of patients with permanent AC Patients’s number Age (years) Serum Vitamin B12 (pg/mL)(normal value = 187-883 pg/mL) Anti-Intrinsic factor Serum gastrin level (pg/mL) 1 58 2000 Positive 2000 2 86 2000 Positive 3000 3 69 405 Positive 3000 4 69 224 Negative 1300 5 80 1642 Positive 3000 6 92 265 Positive 3000 7 68 100 Negative 2100