Standard Reference Strains Research Articles

The role of the C. albicans transcriptional repressor NRG1 during filamentation and disseminated candidiasis is strain dependent.

Candida albicans is one of the most common causes of superficial and invasive fungal diseases in humans. Its ability to cause disease is closely linked to its ability to undergo a morphological transition from budding yeast to filamentous forms (hyphae and pseudohyphae). The extent to which C. albicans strains isolated from patients undergo filamentation varies significantly. In addition, the filamentation phenotypes of mutants involving transcription factors that positively regulate hyphal morphogenesis can also vary from strain to strain. Here, we characterized the virulence, in vitro and in vivo filamentation, and in vitro and in vivo hypha-associated gene expression profiles for four poorly filamenting C. albicans isolates and their corresponding deletion mutants of the repressor of filamentation NRG1. The two most virulent strains, 57055 and 78048, show robust in vivo filamentation but are predominately yeast phase under in vitro hypha induction; the two low-virulence strains (94015 and 78042) do not undergo filamentation well under either condition. In vitro, deletion of NRG1 increases hyphae formation in the SC5314 derivative SN250, but only pseudohyphae are formed in the clinical isolates. Deletion of NRG1 modestly increased the virulence of 78042, which was accompanied by increased expression of hypha-associated genes without an increase in filamentation. Strikingly, deletion of NRG1 in 78048 reduced filamentation in vivo, expression of candidalysin (ECE1), and virulence without dramatically altering establishment of infection. Thus, the function of the conserved repressor NRG1 in C. albicans shows strain-based heterogeneity during infection.IMPORTANCEClinical isolates of the human fungal pathogen Candida albicans show significant variation in their ability to undergo in vitro filamentation and in the function of well-characterized transcriptional regulators of filamentation. Here, we show that Nrg1, a key repressor of filamentation and filament specific gene expression in standard reference strains, has strain-dependent functions, particularly during infection. Most strikingly, loss of NRG1 function can reduce filamentation, hypha-specific gene expression such as the toxin candidalysin, and virulence in some strains. Our data emphasize that the functions of seemingly fundamental and well-conserved transcriptional regulators such as Nrg1 are contextual with respect to both environment and genetic backgrounds.

Genetic Characterization of Wild-Type Measles Viruses Isolated in Peshawar Pakistan

Background: Measles is a highly contagious disease, caused by the Measles Virus (MV). Knowledge of genotypes prevalent in any region is important in effective measles surveillance; by mapping distribution of prevalent MV genotypes at different points of time, in a region, gives an idea about the success of measles control through vaccination programs, by documenting the interruption of transmission of the endemic viral genotype. Objectives: To detect genotypes of wild type measles virus isolated from the samples collected of measles patients in Peshawar valley of Khyber Pakhtunkhwa (KPK) province, Pakistan.Material and Methods: This was a cross-sectional descriptive study. Nasopharyngeal and/or throat swab samples were collected during a measles outbreak, from February to March 2014, from clinically diagnosed measles patients (cases who met the clinical case definition of WHO). Collection and processing were done according to the standard protocols. RNA (Ribo-Nucleic Acid) was extracted directly from samples; cDNA (Complimentary Deoxy-Ribonucleic Acid) was synthesized, amplification and sequencing of 528bp of carboxy-terminal of N gene was carried out. Sequences were analyzed and data were compared with the standard reference strains of measles virus.Results: A total of nine sequences obtained were of good quality and enough in length to meet WHO criteria for genotype assignment. It was found that all the nine strains of measles virus belonged to clade B, genotype B3 and cluster B3.1. The rest of the 13 sequences which were shorter in length for genotype assignment also showed close resemblance to Genotype B3.Conclusion: Wild type measles virus strains, isolated from the samples of measles patients, in Peshawar valley of Khyber Pakhtunkhwa province of Pakistan were found to be genotype B3. It shows that genotype B3 may be the predominate genotype prevalent in Peshawar region at the time of this study.Keywords: B3 genotype, Genotyping of measles, Measles Virus, Molecular epidemiology.

Anti-Quorum Sensing and Anti-Biofilm Activity of Ginger (Zingiber officinale) Rhizomes against Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa.

The aim of this study was to determination of Anti-Quorum Sensing (AQS) and anti-biofilm potential of the methanol extract of ginger (Zingiber officinale) rhizomes against multidrug-resistant clinical isolates of Pseudomonas aeruginosa (P. aeruginosa). The AQS activity of ginger was determined against Chromobacterium violaceum (C. violaceum) ATCC 12472 (CV12472), a biosensor strain, in qualitative manner using the agar well diffusion method. The violacein pigment inhibition was assessed to confirm AQS activity of ginger. The AQS potential of sub-minimum Inhibitory Concentrations (sub-MICs) of the ginger extract was determined by targeting different QS regulated virulence factors, including swarming motility (using swarm diameter measurement method), pyocyanin pigment (using chloroform extraction method), Exopolysaccharide (EPS) (using phenol-sulphuric acid method), and biofilm formation (using microtiter plate assay), against clinical isolates (CIs 2, 3, and 4) and standard reference strain of P. aeruginosa (PA01). The AQS activity of methanol extract of ginger was confirmed against C. violaceum (CV12472) as inhibition of violacein pigment formation without effecting the growth of CIs and PA01 of P. aeruginosa. The ginger extract exhibited concentration-dependent inhibition of virulence factors and biofilm formation. The maximum reduction was found in swarming motility, pyocyanin, EPS and biofilm formation against PA01 (51.38%), CI3 (57.91%), PA01 (63.29%) and CI2 (64.37%), respectively at 1/2 MIC of ginger extract. The results of present study revealed the effective AQS and anti-biofilm potential of Zingiber officinale rhizome methanol extract at a reduced dose (sub-MICs). The extract may be explored as an agent of antimicrobial compounds having AQS and anti-biofilm activity for controlling microbial infection and also for reducing the chances of emergence of resistance in P. aeruginosa.

Identification and Characterization of ten Escherichia coli Strains Encoding Novel Shiga Toxin 2 Subtypes, Stx2n as Well as Stx2j, Stx2m, and Stx2o, in the United States.

The sharing of genome sequences in online data repositories allows for large scale analyses of specific genes or gene families. This can result in the detection of novel gene subtypes as well as the development of improved detection methods. Here, we used publicly available WGS data to detect a novel Stx subtype, Stx2n in two clinical E. coli strains isolated in the USA. During this process, additional Stx2 subtypes were detected; six Stx2j, one Stx2m strain, and one Stx2o, were all analyzed for variability from the originally described subtypes. Complete genome sequences were assembled from short- or long-read sequencing and analyzed for serotype, and ST types. The WGS data from Stx2n- and Stx2o-producing STEC strains were further analyzed for virulence genes pro-phage analysis and phage insertion sites. Nucleotide and amino acid maximum parsimony trees showed expected clustering of the previously described subtypes and a clear separation of the novel Stx2n subtype. WGS data were used to design OMNI PCR primers for the detection of all known stx1 (283 bp amplicon), stx2 (400 bp amplicon), intimin encoded by eae (221 bp amplicon), and stx2f (438 bp amplicon) subtypes. These primers were tested in three different laboratories, using standard reference strains. An analysis of the complete genome sequence showed variability in serogroup, virulence genes, and ST type, and Stx2 pro-phages showed variability in size, gene composition, and phage insertion sites. The strains with Stx2j, Stx2m, Stx2n, and Stx2o showed toxicity to Vero cells. Stx2j carrying strain, 2012C-4221, was induced when grown with sub-inhibitory concentrations of ciprofloxacin, and toxicity was detected. Taken together, these data highlight the need to reinforce genomic surveillance to identify the emergence of potential new Stx2 or Stx1 variants. The importance of this surveillance has a paramount impact on public health. Per our description in this study, we suggest that 2017C-4317 be designated as the Stx2n type-strain.

Minimal Inhibitory Concentrations of Thymol and Carvacrol: Toward a Unified Statistical Approach to Find Common Trends.

Thymol and carvacrol are some of the most important and used components of Essential oils (EOs); they are widely studied, and there are much data available in the literature. Their Minimal Inhibitory Concentration (MIC) values found in the literature from 2005 to present were used to assess the bioactivity toward yeasts, molds, Gram-positive bacteria, and Gram-negative bacteria, as well as on some bacterial species/serotypes (Salmonella sp., Escherichia coli, E. coli O157:H7, lactic acid bacteria, Listeria monocytogenes, Staphylococcus aureus, S. epidermidis, etc.) to find possible common trends or differences between the two compounds and among the tested species. The results were quite interesting and pointed out that there is a common range for the MIC of thymol and carvacrol for some bacterial species (150-400 mg/L), with some exceptions to this generalized statement. In addition, the statistics pointed out that bacteria could experience homogeneous (S. epidermidis, E. coli O157:H7) or heterogeneous trends (for example, Salmonella sp.) depending on the existence of possible sub-species or different experimental set-ups. Moreover, this paper suggests that there are some drawbacks and issues that should be solved for the effective use of EOs, which are the strong variability among the microorganisms and the lack of standard protocols and reference strains.

<i>In vitro</i> Evaluation of Antibiofilm Activity of Methanolic Leaf Extract of <i>Azadirachta indica</i> on Cariogenic <i>Streptococcus mutans</i>

Dental caries is a biofilm-dependent disease, and Streptococcus mutans is the primary etiological agent involved in the initiation of the disease. The extensive use of a limited range of antimicrobial drugs in dentistry has led to the development of drug-resistant bacteria. There is an increasing need to find new alternatives against drug-resistant bacteria. Globally, there is a continuous effort towards identifying natural anti-caries agents for the prevention and better management of caries. The objective of the present study was to evaluate the antibiofilm potential of Azadirachta indica leaf methanolic (ALM) extract against S. mutans biofilm. The study employed a standard reference strain of S. mutans MTCC 497, for in vitro standardisation of biofilm by microtiter plate assay. The antibiofilm activity of the ALM extract was evaluated against the S. mutans strain, and the same was confirmed by light and scanning electron microscopy (SEM). The in vitro biofilm standardisation results demonstrated that 50 µl/ml of S. mutans inoculum concentration exhibited a much superior biofilm formation than the other concentrations employed. Light microscopy and SEM images revealed that ALM extract at 100 mg/ml concentration significantly inhibited the S. mutans biofilm. To conclude, the study reports that the A. indica leaf extract is a potential source to inhibit the S. mutans biofilm. Further studies are warranted to identify the phytochemicals responsible for the antibiofilm activity of ALM extract against S. mutans biofilm that aid in the design of natural anti-caries products.

A Rapid and Sensitive Detection of HIV-1 with a One-Pot Two-Stage Reverse Transcription Recombinase Aided Real-Time PCR Assay.

Human immunodeficiency virus 1 (HIV-1) attacks the immune system, making people susceptible to various diseases, thus increasing their risk of death. Comprehensive detection of major HIV-1 strains circulating in China is vital for effective HIV-1 infection prevention and treatment. HIV-1 nucleic acid detection is considered effective for HIV-1 diagnosis since traditional immunological testing may fail to detect HIV-1 infection during the window period. This work demonstrates a one-pot two-stage amplification assay (RT-RAP), a combination of reverse transcription recombinase (RT- RAA), and quantitative real-time polymerase chain reaction (qRT-PCR). The turn-around time of the assay is only 50 min and can be performed with commonly available laboratory equipment, the qPCR devices. The RT-RAP assay could detect approximately 5 and 14 copies/reaction of HIV-1 DNA and RNA using recombinant plasmids and standard reference strains, respectively. Additionally, we found that the clinical performance of RT-RAP (detected 169 samples out of 170 specimens) was consistent with that of qRT-PCR. The sensitivity and specificity of RT-RAP were 100.00% (99/99) and 98.59% (70/71), respectively, while its positive and negative predictive values were 99.00% (99/100) and 100.00% (70/70), respectively. The total coincidence rate of the RT-RAP was 99.41% (169/170), with a kappa value of 0.988 (p < 0.05). We demonstrated that RT-RAP could rapidly detect the common HIV-1 subtypes commonly circulating in China with comparable sensitivity and specificity to qRT-PCR.

Improved non-redundant species screening panels for benchmarking the performance of new investigational antibacterial candidates against Category A and B priority pathogens.

BackgroundNIAID has a programme for testing drug candidates against biodefense and emerging bacterial pathogens that uses defined strain panels consisting of standard laboratory reference strains and strains of clinical origin.ObjectivesThe current studies were performed to assess the activity of standard-of-care drugs, determine benchmark criteria for new investigational antibacterial candidate prioritization and identify reduced non-redundant strain panels for candidate performance classification.MethodsThe susceptibilities of each strain in the screening panels to 40 standard-of-care drugs and clinical drug combinations were determined by percentage growth inhibition using multiple concentrations, a method commonly used in efficient high-throughput screening efforts. The drug susceptibility of each strain was categorized based on interpretive criteria to benchmark the activity of each standard-of-care drug and drug combination, followed by confirmation of select active drugs. Exact match and clustering analyses defined focused non-redundant species and pan-species screening panels.ResultsThis process revealed a broad spectrum of susceptibilities among strains in each species, with important differences between the standard laboratory reference strains and strains of clinical origin. Exact match and clustering analyses identified subsets of non-redundant strains that can more efficiently classify drug activity resulting in individual species screening panels, a pan-species screening panel and a pan-species maximum resistance panel.ConclusionsThis study resulted in improved non-redundant species screening panels for benchmarking the performance of new investigational antibacterial candidates with the greatest potential for efficacy against clinically relevant Category A and B priority and emerging pathogens.

Establishment of a reference panel of Helicobacter pylori strains for antimicrobial susceptibility testing.

BackgroundEradication treatment for Helicobacter pylori gastritis is covered by national health insurance since 2013 in Japan. However, eradication failure due to the increase of antimicrobial resistance has become a serious problem. The present study aims to establish a reference panel of Japanese H. pylori strains for antimicrobial susceptibility testing.MethodA total of 28 strains were collected from 4 medical facilities in Japan. Antimicrobial susceptibility tests (ASTs) to clarithromycin (CLR), amoxicillin (AMX), and metronidazole (MNZ), were used to select standard reference strains. Complete genome sequences were also determined.ResultsThree H. pylori strains (JSHR3, JSHR6 and JSHR31) were selected as standard reference strains by the Japanese Society for Helicobacter Research (JSHR). The minimum inhibitory concentrations (MICs) of the antibiotics against these 3 strains by agar dilution method with Brucella‐based horse‐serum‐containing agar medium were as follows: JSHR3 (CLR 16 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), JSHR6 (CLR 0.016 μg/ml, AMX 0.032 μg/ml and MNZ 4 μg/ml), and JSHR31 (CLR 16 μg/ml, AMX 1 μg/ml and MNZ 64 μg/ml).ConclusionsA reference panel of H. pylori JSHR strains was established. The panel consisted of JSHR6, which was antibiotic‐susceptible, JSHR3, which was CLR‐resistant, and JSHR31, which was multi‐resistant. This reference panel will be essential for standardized ASTs before the optimal drugs are selected for eradication treatment.

Applicability of rpoB Gene for PCR-RFLP based Discrimination of Bifidobacterial Species Isolated from Human and Animal Sources

Bifidobacteria are widely used as probiotics for their application in the development of functional food and prophylactic therapy. This has necessitated the development of a molecular approach for the genera to be widely identified up to species and subspecies level. In the current study, PCR-RFLP of the partial RNA polymerase β-subunit (rpoB) gene fragment was evaluated for differential identification of Bifidobacterium species. The rpoB gene partial sequences of 575 bp were amplified from 93 previously identified isolates collected from various sources of human and animal origin along with 12 standard reference strains. The PCR amplified products were digested with three restriction endonucleases HhaI, HinfI and BanI separately. Dendrograms constructed from the patterns of HhaI, were found to be more discriminatory and successfully differentiated all the twelve species and also at sub-species level in between B. longum subsp. longum and B. longum subsp. infantis. However, B. adolescentis and B. pseudocatenulatum group clusters were not separated and represented by one group. The groups were further discriminated by HinfI restriction digestion. A separate combination thereof may be used for inferring the classification of bifidobacterial species targeted on rpoB PCR-RFLP analysis. To our knowledge, this work is the first report based on use of rpoB PCR-RFLP for discrimination of the isolates of genus Bifidobacterium and also provides insights into specific advantages of this method over hsp60 PCR-RFLP in differentiating B. longum subsp. longum and B. longum subsp. infantis.

Synthesis and initial testing of novel antimalarial and antitubercular isonicotinohydrazides

Malaria and tuberculosis (TB) though curable and preventable, remain serious public health problems globally, with devastating consequences. Co-infection of these two deadly diseases worsens the situation and particularly makes treatment very difficult. The current mainstay for malaria treatment is gradually losing their potency due to the development of resistance. Macobacterium tuberculosis (MTB) has developed Multi-drug Resistance (MDR) and Extensive Drug Resistance (XDR) to current antitubercular drugs due to patient incompliance resulting from long treatment regimen. A small library of isonicotinohydrazide were synthesised by incorporating 1,2,4,5- tetraoxane and hydrazine moieties. Evaluation of the compounds gave antimalarial activities in the range 0.060 ± 0.033–0.491 ± 0.012 µM against 3D7 strain of Plasmodium falciparum. We assessed antimycobacterial activity of selected compounds against the standard Mycobacterium tuberculosis reference strain H37Rv and M. aurum (a non-tuberculous mycobacteria) using the microplate Alamar blue (MABA) assay and found four compounds to be very potent against H37Rv but largely inactive against Macobacterium aurum. We followed up to estimate the minimum inhibitory concentrations of these active compounds and tested them against clinical M. tuberculosis strains resistant to isoniazid (INH) and rifampicin (RIF). The MICs of the active compounds against H37Rv were between 0.003 and 0.5 mg/mL however, they were largely inactive against drug resistant clinical strains except for the INH mono-resistant resistant strain which was very active with compounds 5 and 8 possessing MICs of 0.125 mg/mL.

ANTIMICROBIAL EFFECT OF CITRATE BUFFER WITH ANTIBIOTIC

Antibiotic resistance of pathogens to medications is an essential problem globally. Thus, new medication compositions are one of the ways to solve this problem. This study aimed to investigate the antimicrobial efficacy of the citrate buffer combined with the antibiotic «Amoxiclav» on standard reference strains of microorganisms. We used standard cultures of C. albicans ATCC10231, E. coli ATCC25922, S. aureus ATCC25923, E. faecalis ATCC29213, M. luteus ATCC4698, S. epidermidis ATCC14990 in the study conducted at the Microbiology, Virology and Immunology Department of the Poltava State Medical University. The sensitivity of standard microorganism strains to the composition was studied with a quantitative method of serial dilutions based on CLSI. ISO/TC 212 «Clinical laboratory testing and in vitro diagnostic test systems. 2021». Thus, the results of this study clearly showed a synergistic effect of citrate buffer and amoxiclav on all the reference strains of microorganisms. Reference strains of Staphylococcus aureus and epidermal staphylococcus showed the highest sensitivity to citrate buffer, which was 4 times (p &lt; 0.05) higher than that shown by micrococci, enterococci and fungi, and 8 times (p &lt; 0.01) higher than that of Escherichia coli, which appeared least sensitive to the test substance. Amoxiclav also inhibited the growth of S. aureus ATCC 25923 and S. epidermidis ATCC 14990 and had the minimal effect on the reference strain of E. coli ATCC 25922, the minimal inhibitory concentration of which was 31.3 times (p &lt; 0.0001) higher than that of staphylococci. The proposed citrate buffer-amoxiclav combination significantly increased the antimicrobial efficacy of its components against all the reference strains of microorganisms. Thus, the sensitivity of E. coli ATCC 25922 and M. lysodeikticus ATCC 4698 to the proposed combination increased 8-fold (p &lt; 0.05) compared to their sensitivity to citrate buffer or amoxiclav alone. The sensitivity of Staphylococcus aureus and enterococcі to the composition increased to the maximum: up to 32-fold (p &lt; 0.001) to the buffer and 4‑fold to the antibiotic (p &lt; 0.01). The effect of a significant increase in the sensitivity of C. albicans ATCC10231 strain to the citrate buffer after adding amoxiclav was gone up 128-fold (p &lt; 0.0001). It was the evidence of a synergistic fungicidal action of the antibiotic-citrate buffer combination.

Extractable surface proteins of indigenous probiotic strains confer anti-adhesion knack and protect against methicillin-resistant Staphylococcus aureus induced epithelial hyperpermeability in HT-29 cell line

Probiotic intervention has been long believed to have beneficial effects on human health by curbing the intestinal colonization of pathogens. However, the application of live probiotics therapy may not be an ideal approach to circumvent the infections of superbug origin due to the risk of horizontal antibiotic resistance genes transfer. In this study, the anti-adhesion ability of extractable cell surface proteins from two indigenous potential probiotic strains (Lactiplantibacillus plantarum A5 and Limosilactobacillus fermentum Lf1) and two standard reference strains (Lactobacillus acidophilus NCFM and Lacticaseibacillus rhamnosus LGG) was evaluated against clinical isolates of Methicillin-Resistant Staphylococcus aureus (MRSA) on porcine gastric mucin and HT-29 cells. The surface proteins from the probiotic strains were extracted by treatment with 5 M lithium chloride. The surface protein quantification and SDS-PAGE profiling indicated that the yield and protein patterns were strain-specific. Surface proteins significantly hampered the mucoadhesion of MRSA isolates via protective, competitive, and displacement. Similarly, the treatment with surface proteins probiotic strains displayed anti-adhesion against MRSA isolates on HT-29 cells without affecting the viability of the cell line. Surface proteins treatment to the confluent monolayer of HT-29 cells maintained the epithelial integrity; however, MRSA isolates (109 cells/mL) showed considerable alteration in the epithelial integrity by exacerbating the FITC-dextran transflux. Contrarily, the co-treatment with surface proteins with MRSA isolates significantly lowered the FITC-dextran transflux across the differentiated HT-29 monolayer. Overall, the findings of this study suggest that probiotic-derived surface proteins could be the novel biotherapeutics to combat the MRSA colonization and their concomitant intestinal infections.

Efficacy of BOX-PCR fingerprinting for taxonomic discrimination of bifidobacterial species isolated from diverse sources.

The genus Bifidobacterium are extensively used as probiotics in food applications, for their potential role to combat different lifestyle diseases. This has necessitated a great importance for their species, sub-species and even at the strain level characterization. In the present study, attempts have been made to target repetitive DNA element-based BOX-PCR fingerprinting to judge its potential in taxonomic discrimination of Bifidobacterium species. The BOXA1R primer-based repetitive PCR amplified products were analysed for 93 identified bifidobacterial isolates collected from diverse sources of human and animal origin along with 12 DSMZ procured standard reference strains. Dendrograms constructed from the fingerprint patterns of BOX-PCR differentiated all the isolated strains into 10 different groups, grouped with one standard reference isolates and successfully discriminated all isolates up to subspecies level as identified. The BOX-PCR method used in this study effectively resolved the taxonomic status and differentiated all 93 bifidobacterial species isolated from diverse faecal origins of human and animal samples.

Antibiotic susceptibility pattern of isolates from urine of students in Benue State Polytechnic, Ugbokolo, Nigeria

Background: Urinary Tract Infections (UTIs) remains one of the most common infectious diseases diagnosed in developing countries. The widespread use of antibiotics against uro-pathogens has led to the emergence of antibiotic resistant species. Objectives: The study aimed at determining the antibiotic susceptibility pattern of isolates from urine of students in Benue State Polytechnic, Ugbokolo, Nigeria. Materials and methods: Three hundred and twenty seven (327) midstream urine samples from 137 (41.9%) males and 190 (58.1%) females were collected from students in hostels of Benue State Polytechnic, Ugbokolo. The urine samples were inoculated into the dried surfaces of Blood agar, MacConkey agar, Eosine Methylene Blue agar and Cysteine Lactose Electrolyte-deficient agar using a calibrated loop. The inoculated plates were aerobically incubated at 37oC for 24h. Colonies were sub-cultured repeatedly to obtain a pure culture. E.coli (ATCC 25922) and S. aureus (ATCC 25923) standard reference strain were used as control. The isolates were identified using cultural and biochemical characteristics such as Gram stain, Triple Sugar Iron agar test, methyl red, Voges-Proskauer, citrate utilization, catalase, oxidase, urease and coaulase test. The identified species were then exposed to selected antibiotics to test for their susceptibility. Results: A high prevalence of 15.9% (n=327) was recorded in the study. Significant differences (P&lt;0.05) were observed in the age, sex and number of isolates. Isolation rate showed female (63.5%, n=33) preponderance over male (36.5%, n=19). Isolation rate was highest in students aged 22-26 (44.2%, n=23). E. coli was the most frequently isolated organism. The Gram negative bacterial isolates showed the highest level of resistance to amoxicillin (92.3%, n=36) and SXT (92.3%, n=36) while the Gram positive exhibited the highest level of resistance to SXT (87.5%, n=7). All the isolates were however susceptible to imipenen and gentamycin. Conclusion: The high prevalence showed by this study suggests the existence of a public health concern among the students. The fact that most of these isolates are resistant to one or more of the commonly used antibiotics calls for con

ANTIMICROBIAL ACTIVITY OF SOME MALAYSIAN MUSHROOMS ON ESCHERICHIA COLI AND STAPHYLOCOCCUS AUREUS

Background and Objective: Nowadays, the world is facing significant challenges in modern healthcare services because many antimicrobial agents have lost their effectiveness in treating infectious diseases primarily due to the development of microbial resistance. Mushroom species release various bioactive compounds and yet largely untapped resource of useful natural compounds. This study aimed to assess the primary screening for antimicrobial activity of selected Malaysian mushroom’s against Escherichia coli and Staphylococcus aureus.Methodology: The mycelia biomass extracts of the 8 collected Basidiomycetes mushrooms. Antibacterial activity was measured using Disc diffusion and Well diffusion methods against standard reference strains E. coli MTCC 740 and S. aureus MTCC 501. Results: The potential inhibition of Agaricus cupreobrunneus, which gave a reading of 4.6 cm for disc diffusion and 3.4 cm for well diffusion of inhibition against E. coli MTCC 740 and against S. aureus MTCC 501 that, gave a reading of 4 cm for disc diffusion and 3.1 cm for well diffusion approaches. The second highest bioactivity mushroom against E. coli MTCC 740 the highest inhibition was shown by extract of brown Agaricus bisporus which gave 3.5 cm and give 3.2 cm S. aureus MTCC 501 by disc diffusion. For well diffusion the zone of inhibition by same mushroom for E. coli MTCC 740 which gave reading 3.0 cm and gave 2.7 cm against S. aureus MTCC 501. Conclusions: The species studied showed potential inhibition of A.cupreobrunneus more than other mushrooms by disc diffusion method especially against E. coli more than S. aureus. Further studies are required to ascertain their toxicity against mammalian cells and potential side effects.

Isolation and characterization of lactic acid bacteria from human milk

Human milk is the main source of nutrition for infants and the transmission of various microorganisms. The lactic acid bacteria (LAB) in breast milk allow for the establishment of the gut microflora of infants. In this study, we aimed to assess the probiotic potential of LAB strains isolated from breast milk of healthy Chinese women. Two strains, Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) LHL6 and LHL7, were selected and identified through morphology observation, Gram staining, and 16S rDNA phylogenetic analysis. Using Limosilactobacillus fermentum (formerly Lactobacillus fermentum) CECT5716 as the standard reference strain, the screened strains were characterized for aspects of growth, production of lactic acid and H2O2, antibiotic susceptibility, survival under simulated gastrointestinal conditions, and tolerance to cadmium (Cd). In de Man, Rogosa, and Sharpe (MRS) broth, LHL6 and LHL7 showed longer lag phases than CECT5716 but higher specific growth rates. For the production of lactic acid and H2O2, LHL7 performed better than LHL6 and CECT5716, indicating better antimicrobial ability. Strain LHL7 generated 9.99 mg/L H2O2, considerably higher than 1.25 mg/L for LHL6 and 2.33 mg/L for CECT5716. According to European Food Safety Authority minimum inhibitory concentrations, all of the investigated strains were resistant to chloramphenicol, streptomycin, and kanamycin. However, unlike LHL6 and CECT5716, LHL7 was susceptible to ampicillin and resistant to tetracycline. Resistance to azithromycin, cephalexin, and penicillin G were similar for all 3 strains, whereas CECT5716 was resistant to a higher concentration of roxithromycin. All 3 strains were able to survive in a simulated gastric-like solution, but a low percentage survived in the presence of 0.4% bile salt and 7% pancreatin. Encapsulation with protectants may enhance the survival rate. All 3 strains were tolerant to 500 mg/L Cd in MRS broth and to 1,000 mg/L Cd on MRS agar medium. In summary, 2 novel strains of LAB were obtained that have similar characteristics to the reference strain CECT5716. This work identified potential probiotic candidates for application in the food and pharmaceutical industries and facilitated identification of further probiotics.

Antioxidant, cytotoxic and antibacterial activity of Rosmarinus officinalis L. essential oil against bacteria isolated from urinary tract infection

IntroductionUrinary tract infection (UTI) is a common infection in humans. The main disadvantage of antibiotic usage for UTI treatment is the emergence of antibiotic-resistant bacteria, which endanger antibiotic efficacy. The essential oil of some plants have antimicrobial activity without enhancing the antibiotic resistance. MethodsIn this study, Rosmarinus officinalis L. from Marivan (Kurdistan Province, Iran) was selected, and essential oil was prepared using a solvent-free microwave extraction method. The essential oil components were evaluated by Gas Chromatography-Mass Spectrometry. The agar diffusion and broth microdilution methods were used to investigate essential oil antibacterial effects against standard reference strains and clinical microbial strains isolated from UTI samples of Al-Zahra hospital in Najaf, Iraq. The antioxidant and cytotoxic activity of the oil was also examined by the β-carotene bleaching assay and brine shrimp lethality test, respectively. ResultsThe main components of the essential oil were 1,8-cineole (17.16 %) and α-pinene (16.95 %). The essential oil showed antibacterial activity against all the tested strains, with inhibition zones and minimum inhibitory concentrations (MIC) in the range of 7.00 ± 0.00–9.6 ± 0.32 mm and 0.06 ± 0.00 to 0.16 ± 0.07 mg/mL, respectively. The minimum bactericidal concentration (MBC) values were equal to MIC values. The greatest microbial inhibitory effect was against Staphylococcus aureus, Klebsiella pneumoniae, and Proteus vulgaris, respectively. Furthermore, the oil indicated good antioxidant capacity and high cytotoxicity. ConclusionThis study demonstrated that R. officinalis L. essential oil from Marivan has noteworthy antibacterial activity and can be useful for the treatment of UTI.

Enabling community-based metrology for wood-degrading fungi

BackgroundLignocellulosic biomass could support a greatly-expanded bioeconomy. Current strategies for using biomass typically rely on single-cell organisms and extensive ancillary equipment to produce precursors for downstream manufacturing processes. Alternative forms of bioproduction based on solid-state fermentation and wood-degrading fungi could enable more direct means of manufacture. However, basic methods for cultivating wood-degrading fungi are often ad hoc and not readily reproducible. Here, we developed standard reference strains, substrates, measurements, and methods sufficient to begin to enable reliable reuse of mycological materials and products in simple laboratory settings.ResultsWe show that a widely-available and globally-regularized consumer product (Pringles™) can support the growth of wood-degrading fungi, and that growth on Pringles™-broth can be correlated with growth on media made from a fully-traceable and compositionally characterized substrate (National Institute of Standards and Technology Reference Material 8492 Eastern Cottonwood Whole Biomass Feedstock). We also establish a Relative Extension Unit (REU) framework that is designed to reduce variation in quantification of radial growth measurements. So enabled, we demonstrate that five laboratories were able to compare measurements of wood-fungus performance via a simple radial extension growth rate assay, and that our REU-based approach reduced variation in reported measurements by up to ~ 75%.ConclusionsReliable reuse of materials, measures, and methods is necessary to enable distributed bioproduction processes that can be adopted at all scales, from local to industrial. Our community-based measurement methods incentivize practitioners to coordinate the reuse of standard materials, methods, strains, and to share information supporting work with wood-degrading fungi.

A cross-sectional study exploring disease characteristics and phylogenetic nature of human cytomegalovirus among infected neonates with congenital nephrotic syndrome.

Congenital nephrotic syndrome (CNS) is a rare but serious condition which affects neonates and is caused by monogenic defects of glomerular structural proteins or congenital viral infections. Several reports have established a causal relationship between human cytomegalovirus (HCMV) intrauterine infection and CNS, but thorough study assessing parameters has not yet been done. This study aimed to ascertain significant demographic, biochemical, serological, inflammatory and etiological parameters with 12months follow-up to clinically identify and monitor neonates with HCMV-associated CNS and sought to decipher the phylogenetic nature of infecting strains. Differences between four patient groups (neonates < 4weeks old) with or without CNS and HCMV infection were compared by unpaired t testing and one-way analysis of variance (ANOVA). Linear regression was performed to assess statistical significance among individual groups. Maximum-likelihood-based phylogenetic analysis was performed with HCMV gH gene sequences to compare clinically isolated and referenced NCBI strains. This was further supported by analysis of effective number of codons (ENc), codon adaptation index (CAI) and mRNA structural variation. Patients with HCMV-associated CNS were found to have significant variations in many studied parameters compared with controls. The majority of clinical strains formed a separate phylogenetic cluster defining them as somewhat distinct from standard reference strains, which was supported by the other analyses. This study defined parameters for monitoring cases of HCMV-associated CNS, which suggest the possible existence of a selection force acting and rendering these HCMV strains able to infect selective host tissues and cause specific disease types.