Antibiotic Susceptibility Assay Research Articles

Identification of Acinetobacter schindleri isolated from Chinese giant salamanders (Andrias davidianus)

At a particular aquaculture facility in Zhangjiajie in China, the Chinese giant salamander (Andrias davidianus) exhibited analogous clinical manifestations, culminating in sequential mortalities. This study used rigorous aseptic sampling methods to isolate bacteria from the affected liver of salamanders to determine the causative agent behind the decline in amphibians. A bacterial pathogen was isolated from diseased A. davidianus, and the strain was named DN-3. This isolate was subjected to bacterial identification, antibiotic susceptibility assays, reinfection experiments, and biochemical profiling. The isolated bacterial strain was definitively identified as Acinetobacter schindleri using 16S rRNA sequence analysis and biochemical identification. Antibiotic susceptibility testing revealed that this isolate was susceptible to neomycin, macrolides, doxycycline, piperacillin, nitrofurantoin, and carbenicillin. Subsequent reinfection assays, in which varying concentrations of the bacterial inoculum were administered to healthy salamanders, confirmed that the pathogen elicited varying degrees of morbidity or mortality within an eight-day observation period. The median lethal dose (LD50) of A. schindleri DN-3 for A. davidianus was calculated to be 6.25×104 CFU/mL. This result supports the significant pathogenicity of the strain for A. davidianus. The findings of this study provide empirical insights into the clinical management and epidemiological control of diseases affecting A. davidianus.

Streptococcus pneumoniae Nasopharyngeal Colonization and Antibiogram of Isolates from Paediatric Population in Enugu, South East Nigeria

Aims: This research aimed to evaluate and identify Streptococcus pneumoniae from nasopharynx samples of children and carry out antibiotic susceptibility assay on the isolates; being that pneumococci is well noted as a significant cause of invasive pneumonia in infants with the challenge of antibiotic resistance, which may worsen due to the emergence of such strains among children population. Methodology: This was a descriptive, cross-sectional study conducted between March and September 2023 in the Nsukka area of Enugu. Samples were collected from 100 children population using sterile swab sticks. These were inoculated unto blood and chocolate agar and then incubated at 37o C, morphological and biochemical characteristics were used for identification. An antimicrobial susceptibility test using the disk diffusion method was performed on isolates. The multi-discs used were pefloxacin 10ug, Gentamicin 10ug, Ampiclox 30ug, Cefuroxime 20ug, Amoxacillin 30ug, Ceftriazone 25ug, Ciprofloxacin 10ug, and Streptomycin 30ug, Co-tirmoxazole 30ug, and, Erythromycin 10ug. Results: The results identified 8.0% of pediatric with pneumococcal carriage. Males were 10.0% while females were 5.0%. The age distributions were as follows: 0-5 years 4(8.7%), 6-10 years 1(3.1%), 11-15 years, 3(13.6%). There was no significant difference in the age range distribution of the pneumococci colonization (p=0.074). There was 66.7% sensitivity rate of S. pneumonia. The resistant rates were 75% each to Ampiclox and Amoxicillin. Cefuroxime was 87.5% resistant.

Probiotic potential of Enterococcus strains with multiple enterocin-encoding genes

In this work, we sought to identify and characterize three strains of Enterococcus isolated from camel milk and fermented wheat, based on the capacity to produce bacteriocins and the probiotic potential. Polymerase chain reaction analyses were used to identify bacterial isolates and structural genes of bacteriocins, and also to detect potential enterococcal virulence genes (cylA, esp, gelE, efaAfs, hyl, ace, asa1, vanA, and vanB). The antimicrobial activity of the strains was investigated in solid media by the agar spot method against several pathogenic bacteria. The probiotic potential of the strains was also analysed using low pH (pH 3.0), bile salt resistance, DNAase, and antibiotic susceptibility assays. The strains were identified using rRNA16S sequencing gene, showing their belonging to E. faecium species. Based on PCR results, E. faecium CM9 and CM18 strains included in the genome the structural gene of enterocin A, enterocin B, and enterocin P, while E. faecium H3 possessed enterocin MR10A/B structural gene. The sequence analysis revealed that the H3 strain included in the genome the structural gene of enterocin L50A/B. All the pathogenic bacteria (Staphylococcus aureus, Salmonella enterica, Listeria monocytogenes, E. coli, and Pseudomonas aeruginosa) were inhibited by the three strains E. faecium CM9, CM18, and H3. In the well diffusion test, the supernatants of the three strains exhibited inhibitory activity against Listeria monocytogenes CECT 4032. The strains showed high tolerance to low pH and bile salts, did not possess DNase, were susceptible to the majority of antibiotics assayed, and did not possess any of the virulence genes analyzed in this study. A promising candidate strains were identified as potential probiotics with anti-bacterial action against pathogenic bacteria.

Pathogenetic characterization of a Micrococcus luteus strain isolated from an infant.

To explore the clinical characteristics of Micrococcus luteus bloodstream infection in an infant and characterize the phenotype and genotype of the isolated strains, as well as seek suitable infection models for assessing virulence. Clinical data was collected from an infant patient diagnosed with M. luteus bloodstream infection. Metagenomic sequencing was performed on the isolated blood sample. The strain was isolated and underwent mass spectrometry, biochemical tests, antibiotic susceptibility assays, and whole-genome sequencing. The Galleria mellonella infection model was used to assess M. luteus virulence. Patient responded poorly to cephalosporins, but recovered after Linezolid treatment. Metagenomic sequencing identified M. luteus as the predominant species in the sample, confirming infection. They were identified as M. luteus with a high confidence level of 98.99% using mass spectrometry. The strain showed positive results for Catalase, Oxidase, and Urea tests, and negative results for Mannose, Xylose, Lactose, Mannitol, Arginine, and Galactose tests, consistent with the biochemical profile of M. luteus reference standards. M. luteus susceptibility to 15 antibiotics was demonstrated and no resistance genes were detected. Predicted virulence genes, including clpB, were associated with metabolic pathways and the type VI secretion system. The infection model demonstrated dose-dependent survival rates. The infant likely developed a bloodstream infection with M. luteus due to compromised immunity. Although the isolated strain is sensitive to cephalosporin antibiotics and has low pathogenicity in infection models, clinical treatment with cephalosporins was ineffective. Linezolid proved to be effective, providing valuable guidance for future clinical management of such rare infections.

IMPACT OF BACTERIAL INFECTIONS IN PERITONEAL DIALYSIS PATIENTS

Bacterial infections can cause significant clinical complications in patients undergoing Peritoneal Dialysis (PD). This study aimed to determine bacterial infection's clinical and psychosocial outcomes in PD patients. A prospective observational study enrolled 150 PD patients through non-probability sequential sampling. Demographic information, duration of PD, previous infections, and laboratory results were collected. Bacterial infections were identified through routine cultures of PD fluids, and antibiotic susceptibility was determined using the disc diffusion method. The average age of PD patients was 52.3 years, with 42% men and 58% women (p<0.05). Staphylococcus aureus infected 22 patients (14.67%), whereas Streptococci infected 18 patients (12%) (p<0.05). Antibiotic susceptibility assays determined that Staphylococcus aureus was 77% sensitive to vancomycin. Seventy-five patients (50%) on PD for more than 19 months encountered infections. Among the infected group, 90 patients (60%) recovered without complications, while 5 (4.2%) died. The study revealed a significant correlation between bacterial infections in PD patients and clinical symptoms, antibiotic resistance patterns, and disease duration. These findings highlight the importance of enhanced patient monitoring and evolving treatment strategies to improve patient outcomes.

Metagenomic investigations on antibiotic resistance and microbial virulence in oil-polluted soils from China.

Engine oil spills have been associated with a wide range of human health problems. However, little is known about the effects of petroleum hydrocarbon pollution on soil microbial communities. In this study, three samples were collected from oil-polluted soils (OPS), and one control soil (CS) from Taolin town, China, near the old engine's scrapes was used. The aims of this study were to conduct metagenomic sequencing and subsequently perform resistome and virulome analysis. We also aimed to validate anti-microbial resistance and virulence genes and anti-bacterial sensitivity profiles among the isolates from oil-polluted soils. The OPS microbial community was dominated by bacterial species compared to the control samples which were dominated by metazoans and other organisms. Secondly, the resistosome and virulome analysis showed that ARGs and virulence factors were higher among OPS microbial communities. Antibiotic susceptibility assay and qPCR analysis for ARGs and virulence factors showed that the oil-polluted soil samples had remarkably enhanced expression of these ARGs and some virulence genes. Our study suggests that oil pollution contributes to shifting microbial communities to more resilient types that could survive the toxicity of oil pollution and subsequently become more resilient in terms of higher resistance and virulence potential.

ANTIPATHOGENIC ACTION AND ANTIBIOTIC SENSITIVITY PATTERN OF THE “BORHANI”-ASSOCIATED LACTIC ACID BACTERIUM WEISSELLA CONFUSA LAB-11

Assessment of the beneficial and safety properties of food-associated microbes is inevitable since they engage in direct interactions with their host via the digestive system. In this view, we have studied the pathogen inhibitory activity and antibiotic susceptibility pattern of a newly isolated lactic acid bacterium obtained from the traditional beneficial beverage borhani. 16S rRNA gene based taxonomic and phylogenetic analysis combined with sugar fermentation tests identified the isolate as Weissella confusa; strain LAB-11. Antimicrobial activity of the lactic acid bacterium was examined using its culture supernatant against ten bacterial pathogens by agar diffusion technique. The isolate inhibited species of Acinetobacter, Bacillus, Escherichia, Klebsiella, Pseudomonas, Salmonella, Shigella and Staphylococcus which indicated a broad spectrum of its antimicrobial activity. Further investigation by coincubation assay revealed a prolonged effect of the antibacterial activity against the above pathogens. The inhibitory activity was found highly effective on the fungal pathogen Candida albicans as well. Antibiotic susceptibility assay revealed that the isolate was sensitive to most of the tested antibiotics while resistant against four antibiotics including ciprofloxacin, cloxacillin, ofloxacin, and vancomycin that might be ascribed to intrinsic resistance. The ability to inhibit a wide range of pathogens while itself being relatively safe concerning the transfer of antibiotic resistance suggests that dairy beverages like borhani which carry such beneficial lactic acid bacteria can be of particular benefits to the consumers exerting preventive effects on associated diseases.

3D bioprinting of E. coli MG1655 biofilms on human lung epithelial cells for building complex in vitro infection models

Biofilm-associated infections are causing over half a million deaths each year, raising the requirement for innovative therapeutic approaches. For developing novel therapeutics against bacterial biofilm infections, complex in vitro models that allow to study drug effects on both pathogens and host cells as well as their interaction under controlled, physiologically relevant conditions appear as highly desirable. Nonetheless, building such models is quite challenging because (1) rapid bacterial growth and release of virulence factors may lead to premature host cell death and (2) maintaining the biofilm status under suitable co-culture requires a highly controlled environment. To approach that problem, we chose 3D bioprinting. However, printing living bacterial biofilms in defined shapes on human cell models, requires bioinks with very specific properties. Hence, this work aims to develop a 3D bioprinting biofilm method to build robust in vitro infection models. Based on rheology, printability and bacterial growth, a bioink containing 3% gelatin and 1% alginate in Luria-Bertani-medium was found optimal for Escherichia coli MG1655 biofilms. Biofilm properties were maintained after printing, as shown visually via microscopy techniques as well as in antibiotic susceptibility assays. Metabolic profile analysis of bioprinted biofilms showed high similarity to native biofilms. After printing on human bronchial epithelial cells (Calu-3), the shape of printed biofilms was maintained even after dissolution of non-crosslinked bioink, while no cytotoxicity was observed over 24 h. Therefore, the approach presented here may provide a platform for building complex in vitro infection models comprising bacterial biofilms and human host cells.

Characteristics of phenotypic antibiotic resistance of Helicobacter pylori and its correlation with genotypic antibiotic resistance: A retrospective study in Ningxia

Geographic differences exist in the antibiotic resistance patterns of Helicobacter pylori. Personalized treatment regimens based on local or individual resistance data are essential. We evaluated the current status of H.pylori resistance in Ningxia, analyzed resistance-related factors, and assessed the concordance of phenotypic and genotypic resistance. Strains were isolated from the gastric mucosa of patients infected with H.pylori in Ningxia and relevant clinical information was collected. Phenotypic antibiotic susceptibility assays (Kirby-Bauer disk diffusion) and antibiotic resistance gene detection (Sanger sequencing) were performed. We isolated 1955 H.pylori strains. The resistance rates of H.pylori to amoxicillin, levofloxacin, clarithromycin, and metronidazole were 0.9%, 42.4%, 40.4%, and 94.2%, respectively. Only five tetracycline-resistant and one furazolidone-resistant strain were identified. Overall, 3.3% of the strains were sensitive to all six antibiotics. Multidrug-resistant strains accounted for 22.9%, of which less than 20% were from Wuzhong. Strains isolated from women and patients with nonulcerative disease had higher rates of resistance to levofloxacin and clarithromycin. Higher rates of resistance to metronidazole, levofloxacin, and clarithromycin were observed in the older age group than in the younger age group. The kappa coefficients of phenotypic resistance and genotypic resistance for levofloxacin and clarithromycin were 0.830 and 0.809, respectively, whereas the remaining antibiotics showed poor agreement. H.pylori antibiotic resistance is severe in Ningxia. Therefore, furazolidone, amoxicillin, and tetracycline are better choices for the empirical therapy of H.pylori infection in this region. Host sex, age, and the presence of ulcerative diseases may affect antibiotic resistance of the bacteria. Personalized therapy based on genetic testing for levofloxacin and clarithromycin resistance may be a future direction for the eradication therapy of H.pylori infection in Ningxia.

Antibiotic sensitivity pattern of uropathogens among diabetic and non-diabetic pregnant women in Dhaka, Bangladesh

Objective: To determine the urinary tract infections (UTIs)-causing bacterial profile and antibiotic susceptibility pattern for the proper and complete treatment of UTIs in pregnant women with diabetes mellitus (DM). Methods: We collected 300 urine samples from diabetic and nondiabetic pregnant women at various gestational ages. After isolating and identifying uropathogens, we conducted an antibiotic sensitivity assay against fourteen commonly used antibiotics: amikacin, amoxicillin, ampicillin, azithromycin, cefixime, ceftazidime, ceftriaxone, cefuroxime, ciprofloxacin, co-trimoxazole, gentamicin, levofloxacin, netilmicin, nitrofurantoin. Results: Among the participants, 70% had DM but only 37.3% had positive uropathogen growth. Five gram-negative bacteria (Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., and Pseudomonas spp.) and 3 gram-positive bacteria (Streptococcus spp., Staphylococcus spp. and Enterococcus spp.) were indentified. Escherichia coli was the most prevalent bacteria (57.1%), followed by Klebsiella spp. (19.6%). In the antibiotic susceptibility assay, we found 96.5% of the isolated organisms were highly susceptible to levofloxacin, 94.2% to netilmicin, 88.2% to nitrofurantoin, 85.1% to amikacin, 79.8% to gentamicin, respectively. On the other hand, 64.8%, 63.6%, 61.8% of the isolates demonstrated high-level resistance to ampicillin, ceftazidime and amoxicillin whereas 38.0%, 37.1%, 33.6%, 30.9% and 30.0% of the organisms were resistant to co-trimoxazole, azithromycin, ciprofloxacin, cefuroxime, cefixime, respectivey. Conclusions: Our results suggest that amikacin, levofloxacin, netilmicin, nitrofurantoin, and gentamycin can be used as first-line treatments for UTIs, whether the patient has DM or not. Contrarily, amoxicillin, ampicillin, ceftazidime, azithromycin, and co-trimoxazole should be avoided in treating UTIs.

Enhance antibiotic resistance and human health risks in aerosols during the COVID-19 pandemic

Aerosols are an important route for the transmission of antibiotic resistance genes (ARGs). Since the 2019 (COVID-19) pandemic, the large-scale use of disinfectants has effectively prevented the spread of environmental microorganisms, but studies regarding the antibiotic resistance of airborne bacteria remain limited. This study focused on four functional urban areas (commercial areas, educational areas, residential areas and wastewater treatment plant) to study the variations in ARG abundances, bacterial community structures and risks to human health during the COVID-19 pandemic in aerosol. The results indicated the abundance of ARGs during the COVID-19 period were up to approximately 13-fold greater than before the COVID-19 period. Large-scale disinfection resulted in a decrease in total bacterial abundance. However, chlorine-resistant bacteria tended to be survived. Among the four functional areas, the diversity and abundance of aerosol bacteria were highest in commercial aera. Antibiotic susceptibility assays suggested elevated resistance of isolated bacteria to several tested antibiotics due to disinfection exposure. The potential exposure risks of ARGs to human health were 2 times higher than before the COVID-19 pandemic, and respiratory intake was the main exposure route. The results highlighted the elevated antibiotic resistance of bacteria in aerosols that were exposed to disinfectants after the COVID-19 pandemic. This study provides theoretical guidance for the rational use of disinfectants and control of antimicrobial resistance.

Isolation of heavy metal-resistant Staphylococcus epidermidis strain TWSL_22 and evaluation of heavy metal bioremediation potential of recombinant E. coli cloned with isolated cadD.

A heavy metal-resistant bacterial strain, TWSL_22 was isolated from an industrial effluent sample and tested for heavy metal tolerance and resistance. The strain was molecularly characterized as Staphylococcus epidermidis based on 16S rDNA gene analysis and the sequence was deposited in the NCBI repository (accession number KT184893.1). Metal removal activity (P<.001) of TWSL_22 was 99.99±0.001%, 74.43±2.51%, and 51.16±4.17% for Cd, Pb, and Cu, respectively. Highest MIC was observed for Cd. Antibiotic susceptibility assays revealed the strain TWSL_22 to be resistant to several antibiotics. The strain was screened for possible heavy metal-resistant genes and presence of cadA, copA, and cadD was confirmed by PCR. A DNA fragment containing complete sequence of cadD (618bp) was isolated and cloned into pET 21a(+), transformed into E. coli BL21 and designated as E. coli/cadDET. E. coli/cadDET showed high metal tolerance capacity and could remove over 82% of heavy metals (Zn2+, Cd2+, Cu2+, and Cr3+) in the industrial effluent.

Identification of pathogens and detection of antibiotic susceptibility at single-cell resolution by Raman spectroscopy combined with machine learning.

Rapid, accurate, and label-free detection of pathogenic bacteria and antibiotic resistance at single-cell resolution is a technological challenge for clinical diagnosis. Overcoming the cumbersome culture process of pathogenic bacteria and time-consuming antibiotic susceptibility assays will significantly benefit early diagnosis and optimize the use of antibiotics in clinics. Raman spectroscopy can collect molecular fingerprints of pathogenic bacteria in a label-free and culture-independent manner, which is suitable for pathogen diagnosis at single-cell resolution. Here, we report a method based on Raman spectroscopy combined with machine learning to rapidly and accurately identify pathogenic bacteria and detect antibiotic resistance at single-cell resolution. Our results show that the average accuracy of identification of 12 species of common pathogenic bacteria by the machine learning method is 90.73 ± 9.72%. Antibiotic-sensitive and antibiotic-resistant strains of Acinetobacter baumannii isolated from hospital patients were distinguished with 99.92 ± 0.06% accuracy using the machine learning model. Meanwhile, we found that sensitive strains had a higher nucleic acid/protein ratio and antibiotic-resistant strains possessed abundant amide II structures in proteins. This study suggests that Raman spectroscopy is a promising method for rapidly identifying pathogens and detecting their antibiotic susceptibility.

First Report on Genome Analysis and Pathogenicity of Vibrio tubiashii FP17 from Farmed Ivory Shell (Babylonia areolata)

Ivory shell (Babylonia areolata) is a commercially important aquaculture species mainly found on the southeast coast of China. However, it has been greatly affected by vibriosis in recent years. In this study, FP17 (a potential pathogen) was isolated from a dying ivory shell with “acute death syndrome” and confirmed as a pathogen via infectious experiment. Furthermore, phylogenetic analysis based on the average nucleotide identity (ANI) sequencing of the 16S rRNA gene and housekeeping genes (ftsz, gapA, gyrB, mreB, pyrH, rpoA, and topA) indicated that FP17 was identical to Vibrio tubiashii. Transmission electron microscopy showed that FP17 is curved and has a short rod shape, with a single flagellum. Besides, the calculated LD50 after the intramuscular injection of FP17 was 2.11 × 106 CFU/g at 14 d. The genome of the FP17 strain consists of two chromosomes and one plasmid with 5,261,336 bp and 45.08% GC content, including 4824 open reading frames (ORFs) and 150 non-coding RNAs (ncRNA). Genome mining revealed that 120 candidate gene clusters, including vibrioferrin and flagellum-related proteins, are responsible for virulence. Comparative genomic analysis showed that vibrioferrin genes, such as pvs and type Ⅵ secretion system protein genes (vas), are specific in V. tubiashii FP17 but not in the ATCC19109 strain. Furthermore, 92 antimicrobial resistance (AMR) genes, such as tufA, tet(35), crp, etc., were mapped within the genome as the potential candidate for virulence, consistent with antibiotic susceptibility assay. This is the first study to describe the complete genome sequence of V. tubiashii infecting ivory shell. The genetic characteristics, virulence factors, and antimicrobial resistance of the V. tubiashii strain FP17 were also explored.

Starter inoculums assisted fermentation of Puntius sp.-Role of Lactiplantibacillus plantarum and staphylococcus piscifermentans to reduce fermentation time while increasing safety.

To evaluate the role of starter inoculums to reduce total fermentation time as well as to improve safety and quality of Sheedal, a fermented fish-product of India. Lactiplantibacillus plantarum and Staphylococcus piscifermentans were isolated from the Sheedal and used as starter inoculums. To justify fermenting and probiotic properties of the starter inoculums, characteristics like proteolytic and lipolytic activity, cell-surface hydrophobicity, cell auto-aggregation and co-aggregation, antibiotic susceptibility and antimicrobial assay were performed. Changes in the count of lactic acid bacteria, staphylococci and Enterobacteriaceae were studied periodically. A higher increase in α-amino nitrogen and TCA-soluble peptides during fermentation was observed in treatment which was inoculated with both L. plantarum and S. piscifermentans. Poor competitiveness of staphylococci in presence of LAB was found. Inoculation of both L. plantarum and S. piscifermentans as starter showed a positive effect to reduce significantly the fermentation period from usual 4-5months to 75 days, whereas, same was achieved after 105th and 120th by the use of either L. plantarum or S. piscifermentans respectively. Reduction of the total fermentation period is considered as an additional advantage in Sheedal technology in addition to the safety of the product as evidenced by low pH and high titratable acidity.

The Probiotic Attributes and Anti-pseudorabies Virus Evaluation of Lactobacillus Isolates.

The emergence of pseudorabies virus (PRV) variants brings serious harm to the swine industry, and its effective treatments are limited at present. As one of the probiotics, the Lactobacillus species have beneficial characteristics of regulating the balance of intestinal flora, inhibiting the growth of pathogenic bacteria and viruses' proliferation, and improving self-immunity. In this study, Lactobacillus plantarum HN-11 and Lactobacillus casei HN-12 were selected and identified through morphology observation, Gram stain microscopy, 16S rRNA sequencing analysis, and specific amplification of the recA gene and pheS gene. All tested isolates exhibited rapid adaptation to the different conditions, excellent acid, and bile tolerance, and sensitivity to Salmonella, Staphylococcus aureus, and Escherichia coli. The antibiotic susceptibility assay displayed the isolates sensitive to most antibiotics and resistant to Lincomycin and Norfloxacin. Moreover, the supernatants of HN-11 and HN-12 inhibited PRV proliferation in ST cells. The results of animal experiments showed that supplementing the challenged mice with the supernatants of Lactobacillus isolates in advance delayed the course of the disease. PRV was detected in the heart, liver, spleen, lung, kidney, and brain tissues of dead mice in the test groups, and its copies in the lungs were significantly decreased compared with the control mice (P < 0.05). These findings proved the advantages of L. plantarum and L. casei as potential probiotic cultures, which could provide a basis for its application in microecological preparations and functional formulations.

Molecular identification, whole-cell protein profiling, antibiotics sensitivity and pathogenicity of Chryseobacterium gleum recovered from diseased fishes of freshwater wetlands in India

Globally, bacterial gill disease is one of the most prominent bacterial diseases affecting both marine and freshwater fishes. This study aimed to investigate diseased Labeo rohita, Labeo catla and Oreochromis niloticus with gill discoloration, tail rot and skin ulcer that resembles the typical sign of bacterial gill disease from East Kolkata Wetlands, India. The results revealed that four isolates of Chryseobacterium gleum were the responsible pathogens. The isolates CIFRI-RRM3, CIFRI-BFG2, CIFRI-SRM4 and CIFRI-TRM5 were identified using bacterial colony morphology, biochemical test, whole-cell protein profiling, 16S rRNA gene amplification and nucleotide sequence analysis. The antibiotic susceptibility assay showed that all the isolates were completely resistant to ampicillin, amoxyclav, cefalexin, cephalothin, ceftriaxone, ceftazidime, cefotaxime, cefuroxime, cefadroxil, chloramphenicol, kanamycin, netilmicin, penicillin-G, ticarcillin, tobramycin, erythromycin and tetracycline. A challenge experiment was also conducted for four isolates with intraperitoneal injection at a dose of 2.5 × 1010 CFU mL−1 to 4.2 × 1010 CFU mL−1in respective fish hosts. Only three isolates (CIFRI-BFG2, CIFRI-RRM3 and CIFRI-TRM5) proved their virulence by producing diseases with mild symptoms of skin lesions, gill disease and cumulative mortality of 10–20%. To the best of our knowledge, this is the first description of the phenotypic, molecular and whole-cell protein profiling of C. gleum derived from freshwater fishes in India.

Screening for Antibacterial Activity of Chemical Extracts from Puerto Rican Plants.

Antibiotic resistance is one of the world’s most urgent public health threats. According to the Centers for Disease Control and Prevention, at least 2.8 million people get an antibiotic-resistant infection each year, and more than 35,000 people die in the United States from these infections. The development of new antibiotics is essential to combat this crisis and prevent the loss of additional lives. Natural products have historically been an essential source for the discovery of novel antibacterial agents. Furthermore, several studies have shown Puerto Rican plant extracts possess biologically active compounds. Therefore, we sought to determine whether extracts derived from Puerto Rican plants have broad-spectrum antimicrobial activity against a variety of pathogenic bacteria. Using a broth microdilution assay, we screened thirteen extracts generated from different plant species for antibacterial activity. Each extract was tested against the ESKAPE pathogens, Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp, which are known to exhibit multidrug resistance. All extracts tested thus far were ineffective at inhibiting bacterial growth with the exception of the Bucida buceras leaf extract which showed broad spectrum antibacterial activity against E. faecalis, K. pneumoniae, and E. aerogenes. We are currently validating the antimicrobial activity of B. buceras extract through other antibiotic susceptibility assays. Here, we have shown that Puerto Rican plants have potential as a source of novel antimicrobial agents.

Antibiotic Resistance and Molecular Profiling of the Clinical Isolates of Staphylococcus aureus Causing Bovine Mastitis from India.

Staphylococcus aureus is an opportunistic bacterium known to cause severe infections in humans and animals. It is one of the major bacteria causing subclinical and clinical mastitis, leading to significant economic losses in livestock industry. In this study, we have isolated and characterized 80 S. aureus clinical isolates from mastitis-infected animals. The analysis of antimicrobial susceptibility, molecular typing, biofilm production and genetic determinants was performed to understand molecular and phenotypic features of the prevalent pathogen. Our antibiotic susceptibility assays showed the majority (57.5%) of isolates to be multidrug-resistant (MDR), 38.75% resistant and 3.75% sensitive. We found 25% isolates to be methicillin-resistant S. aureus (MRSA) based on oxacillin susceptibility assays. In the MRSA group, maximum isolates (95%) were MDR compared to 45% in MSSA. Multilocus sequence typing (MLST) revealed 15 different STs; ST-97 was the most common ST, followed by ST-2459, ST-1, ST-9 and ST-72. The agr typing showed agr-I as the most common type, followed by type II and III. Most isolates developed biofilms, which ranged in intensity from strong to weak. The presence or absence of lukS, a virulence-related gene, was found to have a substantial relationship with the biofilm phenotype. However, no significant association was found between biofilm formation and antimicrobial resistance or other virulence genes. We also found four MRSA isolates that were mecA negative based on molecular assays. Our findings reveal the prevalence of multidrug-resistant S. aureus clinical isolates in India that are biofilm positive and have critical genetic factors for disease pathogenesis causing bovine mastitis. This study emphasizes the need for the comprehensive surveillance of S. aureus and other mastitis-causing pathogens to control the disease effectively.

Drug Resistance and Epigenetic Modulatory Potential of Epigallocatechin-3-Gallate Against Staphylococcus aureus.

Antimicrobial resistance of human pathogens, such as methicillin-resistant Staphylococcus aureus, is described by the World Health Organization as a health global challenge and efforts must be made for the discovery of new effective and safe compounds. This work aims to evaluate epigallocatechin-3-gallate (EGCG) epigenetic and modulatory drug potential against S. aureus in vitro and in vivo. S. aureus strains were isolated from commensal flora of healthy volunteers. Antibiotic susceptibility and synergistic assay were assessed through disk diffusion accordingly to EUCAST guidelines with and without co-exposure to EGCG at final concentrations of 250µg/ml, 100µg/ml, 50µg/ml, and 25µg/ml. Transcriptional expression of orfx, spdC, and WalKR was performed through qRT-PCR. A 90-day interventional study was performed with daily consumption of 225mg of EGCG. Obtained data revealed a high prevalence of S. aureus colonization in healthcare workers and clearly demonstrated the antimicrobial and synergistic potential of EGCG as well as divergent resistant phenotypes associated with altered transcriptional expression of epigenetic and drug response modulators genes. Here, we demonstrate the potential of EGCG for antimicrobial treatment and/or therapeutic adjuvant against antibiotic-resistant microorganisms and report divergent patterns of epigenetic modulators expression associated with phenotypic resistance profiles.