A Comparative Study of Static Biofilm Formation and Antibiotic Resistant Pattern between Environmental and Clinical Isolate of <i>Pseudomonas aeruginosa</i>

Biofilms are dense bacterial colonies, derived from microbially derived sessile community, networked within a polysaccharide matrix with a distinct architecture that has the attachment potential to both alive and abiotic surfaces. Pseudomonas aeruginosa is a model biofilm forming microorganism associated with remarkable morbidity and mortality rate due to emergence of antibiotic resistant pathogenic bacteria. Moreover, Pseudomonas aeruginosa originating from a biofilm is more resistant to a wide range of antibiotics than the planktonic bacteria. This research was planned to develop a comparative study of the biofilm production between potential, antimicrobial resistance of Pseudomonas aeruginosa isolated from mature environmental biofilm and clinical strain of the same species that did not derive from biofilm. It was observed that the Pseudomonas aeruginosa from environmental isolates were resistant to 15 prominent antibiotics, while clinical strain was comparatively resistant to only few of them. A confirmatory analysis of biofilm formation and antibiotic resistance pattern of these two groups of organisms was checked by 96-well microtiter plate and the disc diffusion method respectively. Finally, the results portrayed that the environmental strains with high drug resistance, potentially formed a considerable amount of biofilm in the period of a week whereas;clinical stains formed a negligible amount of biofilm within the same time frame.

In vitro investigation of HBV clinical isolates from Chinese patients reveals that genotype C isolates possess higher infectivity than genotype B isolates

Hepatitis B virus(HBV)genotype B and C are two major genotypes that are prevalent in Asia and differ in natural history and disease progression.The impact of HBV genotypes on viral replication and protein expression has been explored by the transfection of hepatoma cells with replication-competent HBV DNA,which mimics the later stages of the viral life cycle.However,the influence of HBV genotypes on the early events of viral infection remains undetermined,mainly due to the difficulties in obtaining sufficient infectious viral particles for infection assays.Here,we report that a high-titer HBV inoculum can be generated from the transient transfection-based cell model after optimizing transfection conditions and modifying the HBV-expressing construct.By performing in vitro infection assays using transiently transfected derived viruses,we found that clinical genotype C isolates possessed higher infectivity than genotype B isolates.Moreover,we identified a naturally occurring mutation sL21S in small hepatitis B surface protein,which markedly decreased the infectivity of HBV genotype C isolates,but not that of genotype B isolates.In summary,using infectious viral particles provided by the optimized transient transfection-based cell model,we have been able to investigate a wide range of HBV variants on viral infectivity,which may contribute to our understanding of the reasons for different clinical outcomes in HBV infections and the development of therapeutic drugs targeting the early stages of HBV life cycle.

Colorimetric Assay to Determine In Vitro Antibacterial Activity Against Clinical Isolates： Enhanced Activity in Damaged Chinese Masson Pine Needles

A colorimetric assay for antibacterial susceptibility testing of clinical isolates （Escherichia coli, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Bacillus cereus, and Streptococcus pneumoniae） is described based on the reduction of a novel tetrazolium salt, 3-（4,5-dimethylthiazol-2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2Htetrazolium （MTS）, in the presence of phenazine methosulfate （PMS） as an electron-coupling agent. The combination of 200 μg/mL MTS with 25 μmol/L PMS resulted in production of large amounts of formazan within 1 h of exposure. In this setting, fractions extracted from Chinese Masson pine （Pinus massoniana Lamb.） needles damaged by the pine caterpillar Dendrolimus punctatus Walker were found to have enhanced levels of antibacterial activity. These fractions, which were designated ＂Master＂, ＂Technique＂, and ＂Strength＂, were isolated and identified by reverse-phase C18 cartridge concentration, gel filtration, and affinity chromatography. Two fractions purified from healthy and undamaged needles were designated H1 and H2, respectively. For all test bacteria species. Technique produced the lowest minimal inhibitory concentrations （MICs）, ranging from 2 to 32 μg/mL, and H2 produced the highest values, with four of the six M ICs being higher than 128 μg/mL. We found that the Rmax model fitted the data well in that the r^2 ranged between 0.87 and 0.96 （median, 0.92） and no statistically significant deviations from the model were found （P= 0.23）. The median coefficient of variation of the log RC50 values and the slope m of the fitted model for all six strains among the replicates were 38 and 41%, respectively. In the course of the investigation, the physiological and functional factors involved in pest damage to plants were also explored. In summary, the MTS-PMS colorimetric assay has advantages over existing methods for the examination of antibacterial activity, and could be developed further such that it would be suitable for screening new antibiotic molecules.

Bacterial Resistance in Clinical Isolates of Klebsiella Pneumoniae

Ninety-four percent of 336 strains Klebsiellapneumoniae were resistant to ampicillin，while less than 10％were resistant to cefotaxine and ceftazidine.Among all theaminoglycosides tested,amikacin had the strongest activity on K.

Characterization of Class 1 Integron Gene Cassettes among Clinical Bacteria Isolated from One Large Hospital in Northern China

The class 1 integron and complex gene cassettes among different species of clinical isolates in northern China were characterized in this study. 383 clinical isolates were obtained from northern China, and class 1 integrons containing gene cassettes widely distributed among gram negative clinical isolates was observed. We find that the class 1 integron showed positive correlation with multidrug resistance phenotype of gram negative bacteria. In addition, we find that isolates belonged to one species harbored different types of gene cassette arrays, while same types of gene cassette arrays were observed in different species of isolates. The diversity of gene cassette arrays among the isolates indicated the complexity of multidrug resistance in clinical isolates in northern China.

Visual pathways involvement in clinically isolated syndrome in children

AIM: To investigate extent and nature of visual pathways involvement in children with clinically isolated syndrome(CIS).METHODS: Forty-seven patients(age 11-17y) with CIS, which later proved to be multiple sclerosis(MS)onset, and 30 controls underwent visual evoked potentials(VEP) investigation within 12 d from the appearance of the first signs of disease. Latency and amplitude of P100 peak were compared with normative data and between groups.RESULTS: In 58% patients, including those without signs of retrobulbar neuritis, significant slowing of conduction along the central visual pathways(P100latency lengthening) is seen. P100 amplitudes drop(signs of axonal damage) are registered less frequently(29% cases).CONCLUSION: The results indicate that visual pathways are often affected in the MS onset; mostly demyelination signs are seen. Despite MRI significance for MS diagnostic, VEPs proved to be still effective in early diagnosis of MS in children.

Establishment of Murine Infection Models with Biological Clones of Dengue Viruses Derived from a Single Clinical Viral Isolate

Dengue virus(DENV)is a single-stranded RNA virus transmitted by mosquitoes in tropical and subtropical regions.It causes dengue fever,dengue hemorrhagic fever and dengue shock syndrome in patients.Each year,390 million people are estimated to be infected by four serotypes of dengue virus,creating a great burden on global public health and local economy.So far,no antiviral drug is available for dengue disease,and the newly licensed vaccine is far from satisfactory.One large obstacle for dengue vaccine and drug development is the lack of suitable small animal models.Although some DENV infection models have been developed,only a small number of viral strains can infect immunodeficient mice.In this study,with biologically cloned viruses from a single clinical isolate,we have established two mouse models of DENV infection,one is severe lethal infection in immunocompromised mice,and the other resembles self-limited disease manifestations in Balb/c mice with transient blockage of type I IFN responses.This study not only offers new small animal models of dengue viral infection,but also provides new viral variants for further investigations on dengue viral pathogenesis.

Advanced approach for antifungal susceptibility and characterization of resistance properties in clinical and environmental isolates of Cryptococcus species complex

Background:Meningitis due to Cryptococcus neoformans/gattii is a fatal infection affecting immunocompromised population worldwide.Amphotericin B(AmB),fluconazole(FLC)and 5-flucytosine are the drugs of choice to treat the infection.We studied antifungal susceptibility pattern of clinical and environmental cryptococcal species using newer approach and analyze their resistant characteristics.Methods:Eighty clinical(54 C.neoformans and 26 C.gattii)and 18 environmental(14 C.neoformans and 4 C.gattii)isolates were subjected to antifungal susceptibility testing by automated(VITEK2C)method.Minimum inhibitory concentrations(MIC)were analyzed statistically.Genomic DNA of FLC resistant isolates was extracted and amplified to detect presence of CnAFR1 gene.Results:C.neoformans showed 1.85%and 21.4%AmB resistance,and 1.85%and 28.5%FLC-resistance,whereas C.gattii showed 25%and 50%FLC-resistance among clinical and environmental isolates respectively.MIC values were significantly(p<0.05)different for the isolates from 2 sources.CnAFR1 gene sequence analysis revealed phylogenetic relationship among the resistant isolates.Conclusions:This pioneering study provides an insight into the sensitivity patterns of clinical and environmental cryptococcal isolates from south India.The recent emergence of AmB-resistance may transpire as a challenge for the clinicians.As the clinical and environmental isolates are phylogenetically evolved from CnAFR1 gene of Filobasidiella neoformans,the resistance is most probably an inherent attribute.This study emphasizes the need for speciation and antifungal susceptibility testing of cryptococcal isolates from clinical sources to institute appropriate antifungal therapy and to reduce the mortality and morbidity.

Magnetic Resonance Imaging in the Acquired Demyelinating Disorders： A Pediatric Cohort Study

Objective： Describing the characteristics of the MRI （magnetic resonance imaging） of patients with ADD （acquired demyelinating disorder） followed in a specialized pediatric clinic. Methods： Descriptive and retrospective study of the MRIs of a pediatric ADD cohort. The included images were acquired in machines with 1.5 or 3T magnets. Low-quality images were excluded. The radiological characteristics of the lesions were described using the 2010 Revised McDonald Diagnostic Criteria regarding localization, contrast enhancement and optic nerve alterations. Results： Twenty-three patients were included （55% female）. The mean age of the first clinical event was 7.7 years. Most common diagnosis was Clinically Isolated Syndrome （35%）, followed by Multiple Sclerosis （30%）, Neuromyelitis Optica （17%） and Acute Disseminated Encephalomyelitis （17%）. Mean time elapsed until diagnosis was 1.8 years. Follow-up MRIs of ADEM patients showed complete or partial resolution of lesions; MS lesions were mostly localized in the brain and four patients had contrast enhancement of lesions in their last available MRI. All NMO （neuromyelitis optica） patients had extensive spinal lesions, and two had optic neuritis. All patients with CIS （clinically isolated syndrome） had focal spinal lesions and evolved with radiologic improvement. Conclusions： ADEM and CIS patients＇ MRIs showed lesion reduction, while MS and NMO patients developed new lesions during follow-up.

Silver nitrate restores susceptibility of clinical multidrug resistant gram-negative and gram-positive bacteria to amikacin in vitro

Silver nitrate could inhibit the clinical multidrug resistant isolates at high concentrations（with minimal inhibitory concentrations（MICs） from 32 μM to 64 μM）. The activities of amikacin in the presence of sub-lethal silver nitrate（15 μM） were tested for the combinational effects against multidrug resistant clinical isolates in vitro. Silver nitrate restored the susceptibility of drug-resistant Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus to amikacin. It lowered the MICs of amikacin from 〉128 μg/mL to（2–16） μg/mL and 32 μg/mL, respectively, and lowered the MICs of amikacin on extended spectrum β-lactamase-producing Pseudomonas aeruginosa and Escherichia coli from（16–32） μg/mL and 16 μg/mL to（〈1–4） μg/mL and 〈1 μg/mL, respectively.

Neurofilament light chain in demyelinating conditions of the central nervous system: a promising biomarker

Neurofilaments are the major structural proteins of the neuronal cytoskeleton and are classified according to molecular weight into heavy,intermediate,and light chains.They are released into the interstitial fluid and cerebrospinal fluid(CSF)as a consequence of axonal damage.In particular,the light chain(NfL)represents the most abundant and soluble subunit and has been demonstrated to be increased in the CSF of patients with inflammatory,degenerative,vascular,or traumatic injuries in correlation with clinical and radiological activity.Similar results have been obtained measuring serum NfL with high-sensitivity single-molecule array,which enables reliable and repeatable measurement of the low NfL concentrations in serum.In particular,CSF and serum NfL values are strongly correlated in patients with multiple sclerosis(MS)and have been demonstrated to be increased in patients with MS and clinically isolated syndromes(CIS)in accordance with clinical and radiological activity.NfL levels increase in patients with a recent relapse and seem to predict cognitive impairment,long-term outcome,and conversion of CIS to MS.The few available data on patients with other demyelinating diseases suggest that NfL levels are also increased in neuromyelitis optica spectrum disorders and related conditions in correlation with attack severity,suggesting that axonal damage may occur in these disorders.We herein report and discuss published data on the role of NfL as a possible predictor of disease activity,clinical outcome and treatment response in patients with demyelinating conditions of the central nervous system.