Standard Molecular Methods Research Articles

Resistance to Fluoroquinolones and Other Antimicrobials in Culture-Positive <i>Salmonella typhi</i> Isolates in Gulbarga, South India

Background: Typhoid fever is a major public health concern in developing countries. The upsurge in the occurrence of bacterial isolates that are resistant to nalidixic acid; with reduced susceptibility to ciprofloxacin in typhoidal Salmonellae constitutes a challenge to the clinician. Methods: In order to better understand the epidemiology of Salmonella infections in South India, Salmonella typhi isolates were screened from various healthcare centers. Salmonella isolates were identified by using standard phenotypic, serological, antibiotic susceptibility and molecular methods. Results: Among a total of 100 S. typhi isolates 9% were found to be multidrug resistant and 30% were nalidixic acid resistant. Isolates with reduced susceptibility to ciprofloxacin displays single base mutations in the gyrA gene. A very low rate of 1% resistance was found to ciprofloxacin. The only one isolate with ciprofloxacin MIC ≥ 4 μg/ml also showed single mutation in the QRDR of the gyrA gene in S. typhi (GenBank accession no. HQ176349-HQ176368). Conclusions: A very low rate of nalidixic acid resistance with reduced susceptibility to ciprofloxacin was observed in comparison to other endemic areas in isolates of S. typhi from Gulbarga, South India, with steadily increasing NAR S. typhi but decreasing MDR isolations over the study period. This is most likely due to an increased use of ciprofloxacin as a first line drug of choice over more traditional antimicrobial agents for the treatment of typhoid fever.

Design and synthesis of novel quinic acid derivatives: invitro cytotoxicity and anticancer effect on glioblastoma.

Aim: Quinic acid (QA) is a cyclic polyol exhibiting anticancer properties on several cancers. However, potential role of QA derivatives against glioblastoma is not well established. Methodology & results: Sixteennovel QA derivatives and QA-16 encapsulated poly (lactic-co-glycolic acid)nanoparticles (QA-16-NPs) were screened for their anti-glioblastoma effect using standard cell and molecular biology methods. Presence of a tertiary hydroxy and silylether groups in the lead compound were identified for the antitumor activity. QA-16 have 90% inhibition with the IC50 of 10.66μM and 28.22μM for LN229 and SNB19, respectively. The induction of apoptosis is faster with the increased fold change of caspase 3/7 and reactive oxygen species. Conclusion: QA-16 and QA-16-NPs shows similar cytotoxicity effect, providing the opportunity to use QA-16 as a potential chemotherapeutic agent.

A novel hybrid DSMC-Fokker Planck algorithm implemented to rarefied gas flows

In this article, a new hybrid approach combining Fokker Planck (FP) and Direct Simulation Monte Carlo (DSMC) is introduced. The FP approach, which is an approximation of the Boltzmann equation, has been recently introduced for modeling rarefied gas flows. Compared to standard molecular methods like DSMC, it has a lower computational cost. However, the FP model may provide erroneous results in modeling some flow features, including vortex region and shock waves. The DSMC approach is accurate enough; yet, it has a high computational cost, especially at low Knudsen/low Mach number conditions. The purpose of this article is to suggest an optimized hybrid algorithm to benefit from high-speed modeling and sufficient accuracy simultaneously. We show a modified form of the gradient length Knudsen number, i.e., KnGL/Kn, must be considered to define the range of accuracy of the FP method. We used KnGL/Kn as a parameter for switching between DSMC and FP. The cavity flow and the nozzle flow are considered for the investigation of the proposed hybrid algorithm. The dependence of the accuracy and computational cost on KnGL/Kn is reported. The results demonstrate that the optimized KnGL/Kn is between 3 and 4, which gives suitable computational cost and acceptable accuracy.

Characterization of Inactivated Influenza Vaccines Used in the Russian National Immunization Program.

Background: today’s standard quality control methods used to control the protein composition of inactivated influenza vaccines only take into account a few key reference components. They do not allow for thorough characterization of protein compositions. As a result, observation of unpredictable variations in major viral constituents and admixtures of cellular proteins within manufactured vaccines that may seriously influence the immunogenicity and safety of such vaccines has become a pressing issue in vaccinology. This study aims at testing a more sophisticated approach for analysis of inactivated split influenza vaccines licensed in the Russian Federation. The formulations under study are the most available on the market and are included in the Russian National Immunization Program. Methods: liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, in combination with label-free protein quantitation via the intensity-based absolute-quantitation (iBAQ) algorithm, as well as a number of standard molecular analysis methods, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and negative-stain transmission electron microscopy (TEM) were applied. Results: the methods implemented were able to identify dozens of viral and host proteins and quantify their relative amounts within the final formulations of different commercially available inactivated split influenza vaccines. Investigation of molecular morphology of the vaccine preparations using TEM revealed typical rosettes of major surface proteins (hemagglutinin and neuraminidase). DLS was used to demonstrate a size distribution of the rosettes and to test the stability of vaccine preparations at increased temperatures. Conclusions: a holistic approach based on modern, highly productive analytical procedures was for the first time applied for a series of different commercially available inactivated split influenza vaccines licensed in Russia. The protocols probed may be suggested for the post-marketing quality control of vaccines. Comparison of different preparations revealed that the Ultrix® and Ultrix® Quadri vaccines produced by pharmaceutical plant FORT LLC and trivalent vaccine Vaxigrip® produced by pharmaceutical company Sanofi Pasteur have well-organized antigen rosettes, they contain fewer admixture quantities of host cell proteins, and demonstrate good correlation among mostly abundant viral proteins detected by different methods.

Distribution of Enterobacteria in Ready-to-Eat Food in Cafeterias and Retail Food Outlets in Benin City: Public Health Implications

Background: The aim of this study was to determine the occurrence and antibiotic susceptibility profile of enterobacteria isolated from ready-to-eat foods within Benin metropolis, Nigeria.Methods: This was a descriptive study of 210 ready-to-eat food samples comprising fried rice, jollof rice, moi-moi, salad, oil beans, non-oil beans, and African salad obtained from roadside food vendors between January and June 2017. Isolation and identification of enterobacteria isolates were carried out using standard bacteriological and molecular methods. Antibiotic susceptibility profile was carried out using the disc diffusion method.Results: The mean mesophilic bacterial count expressed in log10 CFU/g from the ready-to-eat foods ranged from oil beans (4.3±0.52) to African salad (7.2±1.38). Escherichia coli count ranged between oil beans (1.8±0.16) and African salad (4.1±0.10). Salmonella species count ranged from non-oil beans (2.3±0.17) to African salad (5.2±0.09). Significant differences were observed from the population count of the ready-to-eat foods (p < 0.05). Bacterial isolates recovered from the ready-to-eat foods include Pseudomonas aeruginosa, Citrobacter freundii, Enterobacter cloacae, Salmonella enterica, Escherichia coli, Klebsiella oxytoca. The highest occurrence of the bacterial isolates was Escherichia coli 23(41.07%) while the least was Citrobacter freundii 3(5.36%) and Enterobacter cloacae 3(5.36%). The antimicrobial susceptibility profile of the bacterial isolates revealed that all bacterial isolates were 100% resistant to cefepime, ceftazidime, cefuroxime, ertapenem, and meropenem; with considerable sensitivity to kanamycin and gentamycin.Conclusion: The occurrence of these bacterial isolates in the foods constitutes public health risk to consumers as these pathogens have been associated with foodborne infections.
 Keywords: Antibiotic-resistant; Enterobacteria; Foodborne pathogens; Microbial quality; Public health; Street foods

Antibiogram Signatures of Some Enterobacteria Recovered from Irrigation Water and Agricultural Soil in two District Municipalities of South Africa.

This study was undertaken to evaluate the antibiogram fingerprints of some Enterobacteria recovered from irrigation water and agricultural soil in two District Municipalities of the Eastern Cape Province, South Africa using standard culture-based and molecular methods. The prevalent resistance patterns in the isolates follow the order: Salmonella enterica serovar Typhimurium [tetracycline (92.3%), ampicillin (69.2%)]; Enterobacter cloacae [amoxicillin/clavulanic acid (77.6%), ampicillin (84.5%), cefuroxime (81.0%), nitrofurantoin (81%), and tetracycline (80.3%)]; Klebsiella pneumoniae [amoxicillin/clavulanic acid (80.6%), ampicillin (88.9%), and cefuroxime (61.1%)]; and Klebsiella oxytoca [chloramphenicol (52.4%), amoxicillin/clavulanic acid (61.9%), ampicillin (61.9%), and nitrofurantoin (61.9%)]. Antibiotic resistance genes detected include tetC (86%), sulII (86%), and blaAmpC (29%) in Salmonella enterica serovar Typhimurium., tetA (23%), tetB (23%), tetC (12%), sulI (54%), sulII (54%), catII (71%), blaAmpC (86%), blaTEM (43%), and blaPER (17%) in Enterobacter cloacae., tetA (20%), tetC (20%), tetD (10%), sulI (9%), sulII (18%), FOX (11%) and CIT (11%)-type plasmid-mediated AmpC, blaTEM (11%), and blaSHV (5%) in Klebsiella pneumoniae and blaAmpC (18%) in Klebsiella oxytoca. Our findings document the occurrence of some antibiotic-resistant Enterobacteria in irrigation water and agricultural soil in Amathole and Chris Hani District Municipalities, Eastern Cape Province of South Africa, thus serving as a potential threat to food safety.

Many-body reactive force field development for carbon condensation in C/O systems under extreme conditions.

We describe the development of a reactive force field for C/O systems under extreme temperatures and pressures, based on the many-body Chebyshev Interaction Model for Efficient Simulation (ChIMES). The resulting model, which targets carbon condensation under thermodynamic conditions of 6500 K and 2.5 g cm-3, affords a balance between model accuracy, complexity, and training set generation expense. We show that the model recovers much of the accuracy of density functional theory for the prediction of structure, dynamics, and chemistry when applied to dissociative condensed phase systems at 1:1 and 1:2 C:O ratios, as well as molten carbon. Our C/O modeling approach exhibits a 104 increase in efficiency for the same system size (i.e., 128 atoms) and a linear system size scalability over standard quantum molecular dynamics methods, allowing the simulation of significantly larger systems than previously possible. We find that the model captures the condensed-phase reaction-coupled formation of carbon clusters implied by recent experiments, and that this process is susceptible to strong finite size effects. Overall, we find the present ChIMES model to be well suited for studying chemical processes and cluster formation at pressures and temperatures typical of shock waves. We expect that the present C/O modeling paradigm can serve as a template for the development of a broader high pressure-high temperature force-field for condensed phase chemistry in organic materials.

Antibiotic-resistant staphylococci from the wastewater treatment plant and grey-water samples in Obafemi Awolowo University, Ile-Ife, Nigeria.

This study examined the occurrence and molecular basis for antibiotic-resistant staphylococci from the wastewater treatment plant and grey-water samples in Obafemi Awolowo University, Nigeria. Standard microbiological techniques and molecular methods were utilized. The species identified (MALDI score >1.7) comprised S. saprophyticus (19), S. cohnii (8), S. sciuri (7), S. aureus (4), S. epidermidis (3), S. warneri (2), S. equorum (1), S. haemolyticus (1), S. nepalensis (1), S. condimenti (1), and S. pasteuri (1). Resistance to trimethoprim, tetracycline and cefoxitin were observed in 78.3% (47/60), 36.7% (22/60) and 25% (15/60) of the isolates, respectively. The rate of multidrug resistance was 53.3% (32/60) and observed in eight species from different sampling sites. Seven (S. sciuri; n = 5; S. aureus; n = 1; S. warneri; n = 1) of the 20 selected (representing the various staphylococcal species and antibiotypes) isolates were mecA-positive. Furthermore, the tetK gene was detected in nine isolates, six with dfrA, and four were positive for the dfrG gene. One S. aureus was mecA, tetK and dfrG gene positive. The study provides insights on antibiotic-resistant staphylococci from a non-clinical setting and highlights the need for active surveillance to understand the burden of antimicrobial resistance in Nigeria. This is key to improve synergy across the human, animal and environmental health sectors in Nigeria.

Evaluation of three different filters and two methods for recovering viruses from drinking water

Among the enteric viruses implicated in waterborne outbreaks, human norovirus and hepatitis A virus (HAV) are a serious public health issue. Most foodborne viruses are difficult or currently unlikely to cultivate. Because of the lack of a cell culture method, real-time reverse transcriptase PCR is commonly used for the detection of norovirus in foodstuffs and environmental samples. Due to low infectious doses in humans and low virus concentration in water sample, filter adsorption methods were used for concentrating viruses from water. The ISO (Anonymous, ISO 15216-1, 2017) describes standardized molecular methods for detecting HAV and norovirus in bottled water. This method includes a two-step procedure: concentrating the virus using a microporous electropositive filter (47 mm diameter, 0.45 μm pore size) then molecular detection. The Zetapor filter, which had a charged membrane with a pore size of 0.45 μm, was commonly used in the past to concentrate viruses from water or from salad leaves following virus elution. But, unfortunately, the Zetapor filter is no longer marketed and it is therefore necessary to assess an alternative filter. The aim of this study was to compare the ability of two electropositive filters with a pore size of 0.45 μm or 0.22 μm and one uncharged filter (0.45 μm) to recover norovirus and HAV from two different types of drinking water (bottled water and tap water) with the adsorption-elution method proposed by ISO (Anonymous, ISO 15216-1, 2017) (method A) and with direct viral extraction using filters (method B). The mean extraction yields for norovirus and HAV calculated with RNA extracts ranged from 0.2 % – 4.81 % with method A and from 5.05 % – 53.58 % with method B, and did not differ significantly between the two types of drinking water tested. For method B, the mean extraction yields for HAV and norovirus were evaluated according to results from the three filters used. The recovery rate of HAV and norovirus ranged between 3.47 % and 62.41 % with the 0.45 μm electropositive filter and were higher than the other filters. The 0.45 μm electropositive filter could be used to concentrate viruses for routine viral monitoring of drinking water for researchers who want to adopt the method in their lab routine.

Molecular characterization and evaluation of crude oil remediation potential of some rhizobia isolated from plant root nodules

This study aimed to determine the molecular identities and genetic relatedness of rhizobia isolated from pigeon pea and pinto beans, and assess their remediation potential in the presence of 1 %, 3 % and 5 % (w/v) crude oil in minimal medium for 7 days incubation period. Standard microbiological and molecular methods which include amplification and purification of 16S rRNA, agarose gel electrophoresis, and sequencing. Results showed molecular identities of six rhizobia from pigeon peas as Bradyrhizobium diazoefficiens USDA122, Rhizobium leguminosarum WSM2304, Bradyrhizobium japonicum N61, Rhizobium leguminosarum N741, Rhizobium leguminosarum BIHIB1217, and Bradyrhizobium japonicum E109; and three rhizobia obtained from pinto beans were Rhizobium leguminosarum N871, Bradyrhizobium diazoefficiens USDA110 and Bradyrhizobium japonicum SEMIA5079. All tested rhizobia (9) showed petroleum degradation ability, as they all grew in the 1, 3 and 5 % (w/v) crude oil minimal medium under laboratory conditions. B. diazoefficiens USDA122 showed the highest optical density (OD) value of 1.184 ± 0.05 on 7th day at 1 % (w/v) crude oil contamination, while R. leguminosarum N741 has the lowest OD value of 0.372 ± 0.02 at 5 % (w/v) crude oil on 7th day. For all the rhizobia, increase occurred throughout incubation period at 1, 3 and 5 % (w/v) except Rhizobium leguminosarum N741 and R. leguminosarum BIHIB1217. In conclusion, the association of R. leguminosarum BIHIB1217 and R. leguminosarum N871 from pigeon pea and pinto beans respectively, were found most effective in crude oil degradation and thus they are recommended as a promising association for remediation of crude oil spilled soils.

Molecular characterization of Methicillin resistant <I>Staph. aureus </I>from poultry farms in Kano State, Nigeria

The increasing rate of drug resistance associated with Methicillin resistant Staph. aureus is not only a problem in clinical sector but also in livestock disease treatment and management. This study was set out to evaluate the incidence of Methicillin resistant Staph. Aureus from poultry farm birds and the farm workers, and also to evaluate the likelyhood of cross-infection between the birds and the farm workers in Kano State, Nigeria, using standard microbiological and molecular methods. The results showed that out of the 1260 samples collected, 98(33.8%) isolates were confirmed to be Staph. aureus. Using disc diffusion method, the antibiotics susceptibility test results showed that 68 (69.4%) of the isolates were susceptible to cefoxitin while 30 (30.6%) were resistant. While 100% of the isolates were resistant to Ampicillin and Amoxicillin, 93.3% to Oxytetracycline, 90% to Chloramphenicol, 80% to Erythromycin, 76.7% to Oxacillin, 63.3% to Trimethroprim/Sulphamethozaxole, 30% to Ciprofloxacin and 26.7% to Gentamicin. The result also showed that 83.3% of the isolates had multiple antibiotic resistance index (MARI) of > 0.3 and were also multidrug resistant (MDR) while 16.7% had MARI ≤ 0.3. The molecular analysis showed that all the isolates were Staph. aureus of 800bp, 66.7% of the MDR isolates possess MecA gene (162bp), while 33.3% had MecA of500bp. Further analysis showed that 3 of the seven housekeeping genes (pta, gmk and yqil) were also present in the MDR isolates at 43.3%, 20% and 16.7% respectively while 10% express spa typing. The results also showed that there is a correlation between phenotypic cefoxitin resistance and carriage of MecA gene. The implication of these results in cross infection between the farm workers and the poultry birds as well as the danger of transfer of these resistant isolates to the community is discussed in this report.
 Key words: Staph. aureus, cefoxitin resistance, poultry farms, housekeeping genes, spa typing, MDR.

Temporal Appraisal and Molecular Characterization of Escherichia coli from Oyun River, Kwara State, Nigeria

Introduction: Escherichia coli, an indicator of feacal contamination has been proven to be the cause of several disease outbreaks in countries and continents around the world.
 Aim: To determine the genotypic variants of Escherichia coli present in Oyun River and provide information regarding the high-risk variants of E. coli in Oyun River.
 Study Design: The study cuts across the two seasons of Nigeria’s tropical climate weather, being the peak of the Harmattan season and the onset of the Rainy season. Three sampling sites (Jimba Oja; Unilorin Dam and Oyun in Ilorin, Kwara State) along the River course were examined for three months (February – April).
 Methodology: Heterotrophic counts, coli form counts and molecular characterization via PCR using 16 sRNA primers, of water samples were done using standard microbiological and molecular methods.
 Results: Bacteriological results showed monthly mean values of microbial counts, ranged from 23.5 x 106 – 45.17 x 106 cfu/mL and total coli form count ranged from 53 cfu/100 mL to 256 cfu/100 mLs, both of which exceed the WHO standards of 100 cfu/mL for total microbial count and <1 cfu/100 mLs for total coliforms. A total of forty-eight coliforms were isolated, thirty two of which were Escherichia coli. Sequencing and BLAST analysis of eleven of the isolates using NCBI’s online database revealed five different strains. They include: Escherichia coli FAP1 genome (9.1%); Escherichia coli strain ST2747 (54.5%); Escherichia coli strain EADK4 (9.1%); Escherichia coli strain ST540 (18.2%) and Escherichia coli strain NCM3722 (9.1%). Correlation of results with previous studies showed that most of the strains identified were pathogenic. The E. coli strains isolated, coupled with the bacterial load, coliform count and some physicochemical parameters of Oyun River makes it unsafe for public consumption if not treated. Efforts therefore should be made to treat the water before use, while making frantic efforts to prevent further contamination of Oyun River.

In vitro activity of colistin against multidrug-resistant Acinetobacter baumannii isolates harboring blaOXA-23-like and blaOXA-24-like genes: A multicenter based study.

This study was aimed to evaluate occurrence of antibiotic resistance and the presence of resistance determinants among clinical isolates of Acinetobacter baumannii. This cross-sectional study from January to September 2018 was performed on 59 A. baumannii strains isolated from clinical samples in the north of Iran. Isolates were identified by standard microbiologic tests and molecular method. Antimicrobial susceptibility testing was carried out by disk diffusion and broth microdilution methods. The presence of carbapenem resistance genes was detected by PCR method. All isolates were resistant to cefepime, meropenem, imipenem and ceftazidime. The lowest resistance rate was observed against doxycycline with 33.9%. Minimum inhibitory concentration (MIC) results showed that all carbapenem-resistant A. baumannii (CRAB) isolates were susceptible to colistin with MIC50 and MIC90 values of 1/2 µg/mL. Among 59 CRAB, blaOXA-23-like was the most prevalent gene (86.4%) followed by blaOXA-24-like (69.5%). Meanwhile, none of the clinical isolates harbored blaOXA-58-like gene. We found a high prevalence of CRAB strains harboring OXA-type carbapenemases in the north of Iran. Our results suggests that the presence of OXA-type genes was not directly correlated with the increase of imipenem MIC level, but can be clinically important as they contribute to the selection of CRAB strains.

The frequency of virulent genes and antimicrobial resistance patterns of diarrheagenic Escherichia coli isolated from stools of children presenting with diarrhea in a tertiary hospital in Abakaliki, Nigeria

Aim: This study was aimed to determine the virulent genes and antibiotic resistance patterns among circulating diarrheagenic Escherichia coli (DEC) pathotypes in a tertiary care health center in east of Nigeria. Materials and Methods: Diarrheal stool samples were obtained from 80 children under 5 years and E. coli was isolated and identified using standard biochemical and molecular methods. Multiplex polymerase chain reaction (PCR) was used to detect eight virulent genes of DEC. Disk diffusion method was used to determine the antibiotic susceptibility of DEC. Results: DEC infection was observed in 54 (68%) children among which ial gene for enteroinvasive E. coli (EIEC) (40% [n=22]) was commonly detected followed by eltA/eltB for enterotoxigenic E. coli (ETEC) (30% [n=16]), pCVD for enteroaggregative E. coli (EAEC) (20% [n=11]), and eaeA/bfpA for typical enteropathogenic E. coli (EPEC) (10% [n=5]). The DEC isolates phenotypically exhibited resistance for ampicillin (AMP) (44 [81%]), followed by ciprofloxacin (CIP)/ levofloxacin (LEV) (28 [52%]), cefoxitin (FOX) (11 [20%]), and amoxicillin-clavulanic acid (AMC) (6 [11%]). About 60% isolates of stable toxins-ETEC were resistant to AMC, CIP, and LEV while all the labile toxin-ETEC exhibited resistance to AMP. About 60% (n=6) resistance were seen in EAEC against ampicillin, AMC, FOX, CIP, and LEV. In EIEC, all the isolates (n=22) were resistant to AMP while 50% (n=11) were resistant to both CIP and LEV. All EPEC (n=5) were resistant to AMP, FOX, CIP, and LEV. Conclusion: High frequency of virulent ial and eltA/eltB genes for EIEC and ETEC, respectively, suggests that they are the primary etiological agents of diarrhea in children among DEC pathotypes. Resistance of DEC to more than two classes of antibiotics indicate possible emergence of multidrug resistance.

Loop-Mediated Isothermal Amplification (LAMP) for the Diagnosis of Zika Virus: A Review.

The recent outbreak of Zika virus (ZIKV) in the Americas and its devastating developmental and neurological manifestations has prompted the development of field-based diagnostics that are rapid, reliable, handheld, specific, sensitive, and inexpensive. The gold standard molecular method for lab-based diagnosis of ZIKV, from either patient samples or insect vectors, is reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The method, however, is costly and requires lab-based equipment and expertise, which severely limits its use as a point-of-care (POC) tool in resource-poor settings. Moreover, given the lack of antivirals or approved vaccines for ZIKV infection, a POC diagnostic test is urgently needed for the early detection of new outbreaks and to adequately manage patients. Loop-mediated isothermal amplification (LAMP) is a compelling alternative to RT-qPCR for ZIKV and other arboviruses. This low-cost molecular system can be freeze-dried for distribution and exhibits high specificity, sensitivity, and efficiency. A growing body of evidence suggests that LAMP assays can provide greater accessibility to much-needed diagnostics for ZIKV infections, especially in developing countries where the ZIKV is now endemic. This review summarizes the different LAMP methods that have been developed for the virus and summarizes their features, advantages, and limitations.

A comparison between the morphological and molecular approach to identify the benthic diatom community in the St Lucia Estuary, South Africa

The use of diatoms as bioindicators to detect human-induced change is a globally accepted monitoring tool. DNA metabarcoding is an effective complementary tool to standard microscopic methods for species identification, providing reliable and timeous diatom assessments in a range of aquatic ecosystems. This study evaluated the suitability of the DNA metabarcoding approach for diatom monitoring in the St Lucia Estuary, South Africa. Results obtained from the standard microscopic (morphological) and molecular methods were compared and indicated a low similarity in the vegetated (11%), unvegetated (12%) and epiphytic (12%) habitats at species level for the epipelic and epiphytic diatom communities. The difference between the methods was expected given the challenges pertaining to the incomplete molecular reference database, intragenomic variability, physiological differences (number of chloroplasts) and highly variable microphytobenthos biomass. While the molecular method was unable to identify the presence of all the dominant diatom species (routinely used as ecological indicators), the available molecular operational taxonomic units (MOTUs) provided an effective complementary tool to determine the relevant community diversity estimates required for the application of the South African estuarine health index (EHI).

Chronic myeloid leukemia - update 2020

Chronic myeloid leukemia - update 2020 Abstract. The discovery of specific inhibitors of the BCR-ABL tyrosine kinase more than 20 years ago has revolutionized the treatment of patients with chronic myeloid leukemia (CML). Prognosis and outcome of patients considerably improved and progress in the use of the tyrosine kinase inhibitors is ongoing. In comparison to imatinib, second generation inhibitors used in first line lead to faster and deeper molecular remissions accompanied by different adverse event profiles. An essential part of the management of CML patients is to assess regularly the remaining tumor load by standardized molecular methods. Based on several clinical trials definitions of optimal response to treatment and of treatment failure have been put forward and help guide the treatment of the patients. The concept of treatment free remission was investigated extensively and is now part of the management of CML patients. Advanced stages of CML are diagnosed less frequently but are still challenging to treat. In these cases, allogeneic transplantation still plays an important role in the attempt to control the disease.

Survey of Intact and Nonintact Raw Pork Collected at Retail Stores in the Mid-Atlantic Region of the United States for the Seven Regulated Serogroups of Shiga Toxin–Producing Escherichia coli

A total of 514 raw pork samples (395 ground or nonintact and 119 intact samples) were purchased at retail stores in Pennsylvania, Delaware, and New Jersey between July and December 2017. All raw pork samples were screened for serogroup O26, O45, O103, O111, O121, O145, or O157:H7 cells of Shiga toxin-producing Escherichia coli (STEC-7) using standard microbiological and molecular methods. In short, 21 (5.3%) of the 395 ground or nonintact pork samples and 3 (3.4%) of the 119 intact pork samples tested positive via the BAX system real-time PCR assay for the stx and eae virulence genes and for the somatic O antigens for at least one of the STEC-7 serogroups. However, none of these 24 presumptive-positive pork samples subsequently yielded a viable isolate of STEC displaying a STEC-7 serogroup-specific surface antigen in combination with the stx and eae genes. These data suggest that cells of STEC serogroups O26, O45, O103, O111, O121, O145, or O157:H7 are not common in retail raw pork samples in the mid-Atlantic region of the United States.

Improved Species-Level Clinical Identification of Enterobacteriaceae through Broad-Range dnaJ PCR and Sequencing

Enterobacteriaceae represent a diverse and medically important family of bacteria that are difficult to identify to the species level using the standard molecular method of 16S rRNA gene sequencing. Prior work has demonstrated the value of dnaJ gene sequence analysis in resolving different members of the family. However, existing protocols are not optimized for clinical use and exhibit several limitations in practice. Here, we describe an improved assay for dnaJ-based identification of Enterobacteriaceae which boasts increased broad-range specificity across genera, shorter amplicon sizes that are suitable for use with formalin-fixed or direct patient specimens, and enhanced amplification efficiency and assay sensitivity through the incorporation of locked nucleic acid chemistries. Sequence analysis of public databases indicates that the partial dnaJ sequence interrogated by this design retains high discriminatory power among Enterobacteriaceae genera and species, with only particular lineages of Shigella sp. and Escherichia coli proving unresolvable. Limits of detection studies using 8 disparate species indicated that amplification was consistently achievable across organisms and allowed robust dideoxynucleotide chain terminator sequencing from as little as 10 genome equivalents of template, depending on the species interrogated. Retrospective application of the dnaJ assay to patient specimens enabled unambiguous classification of Enterobacteriaceae to the species level in 22 of 27 (81.5%) positive specimens examined, with most remaining cases representing unresolvable calls between closely related Escherichia coli and Shigella species. We expect that this assay will facilitate the accurate molecular identification of species from the Enterobacteriaceae family in a variety of clinical specimens and diagnostic contexts.

Prevalence and molecular epidemiology of Staphylococcus aureus nasal colonization in four nursing home residents in Crete, Greece

Nursing homes are considered as reservoirs for methicillin-resistant Staphylococcus aureus (MRSA). The present study investigated the point prevalence and molecular epidemiology of S. aureus colonization among nursing home residents. The study population comprised of 227 residents, living in four nursing homes of the Heraklion, Crete, Greece area, between January and December 2015. From each nursing home, swabs from the anterior nares of all eligible participants were obtained within a 2-week period. The isolated S. aureus strains were identified and screened by standard microbiological and molecular epidemiological methods. S. aureus carriage was found in 62 out of 227 participants (38.4%) with 33 out of 62 (53.2%) being MRSA. The median age was 83 years (range 52–103). Females were more frequently colonized [47 (75.8%)]. All 33 methicillin resistant Staphylococcus aureus (MRSA) isolates were mecA-positive carrying SCCmec type IV, 30 (91%) the fnbA, and 17 (51.5%) the PVL genes. Thirty-two (97%) belonged to a single pulsotype C; among them, the PVL-positives belonged to ST80 clone, whereas, the PVL-negatives to ST225. Among the 33 MRSA isolates, 32 (97%) were clindamycin-resistant, carrying the ermA gene. Methicillin-susceptible Staphylococcus aureus (MSSA) strains showed polyclonality and 76% were PVL-positive. In conclusion the present study has shown that nursing homes in our area can be regarded as important reservoirs for community-associated MRSA (CA-MRSA).