Phenotypic Identification Methods Research Articles

Molecular insights intoexpression and silencing of resistance determinants in Staphylococcus aureus.

This study aimed to investigate the status of antimicrobial-resistant strains of Staphylococcus aureus in Pakistan, their association in terms of co-occurrence with the biofilm-forming genes, resistance profiling and associated discrepancies in diagnostic methods. A total of 384 milk samples from bovine was collected by using convenient sampling technique and were initially screened for subclinical mastitis, further preceded by isolation and confirmation of S. aureus. The S. aureus isolates were subjected to evaluation of antimicrobial resistance by phenotypic identification using Kirby-Bauer disc diffusion method, while the genotypic estimation was done by polymerase chain reaction to declare isolates as methicillin, beta-lactam, vancomycin, tetracycline, and aminoglycoside resistant S. aureus (MRSA, BRSA, VRSA, TRSA, and ARSA), respectively. The current study revealed an overall prevalence of subclinical mastitis and S. aureus to be 59.11% and 46.69%, respectively. On a phenotypic basis, the prevalence of MRSA, BRSA, VRSA, TRSA, and ARSA was found to be 44.33%, 58.49%, 20.75%, 35.84%, and 30.18%, respectively. The results of PCR analysis showed that 46.80% of the tested isolates were declared as MRSA, 37.09% as BRSA, and 36.36% as VRSA, while the occurrence of TRSA and ARSA was observed in 26.31% and 18.75%, respectively. The current study also reported the existence of biofilm-producing genes (icaA and icaD) in 49.06% and 40.57% isolates, respectively. Lastly, this study also reported a high incidence of discrepancies for both genotypic and phenotypic identification methods of resistance evaluation, with the highest discrepancy ratio for the accA-aphD gene, followed by tetK, vanB, blaZ, and mecA genes. The study concluded that different antibiotic resistance strains of S. aureus are prevalent in study districts with high potential to transmit between human populations. The study also determined that there are multiple resistance determinants and mechanisms that are responsible for the silencing and expression of antibiotic resistance genes.

Gonococci – Pathogens of Growing Importance. Part 1. Current Data on Diagnostics, Genotyping and Therapy.

Abstract Neisseria gonorrhoeae is an aetiological agent of gonorrhoea, which is a sexually transmitted disease (STD), a public health challenge worldwide. Gonorrhoea is undoubtedly a serious health risk, especially for women, due to its often asymptomatic course and the possibility of upper genital tract complications such as pelvic inflammatory disease (PID), which can result in infertility. The data show that the diagnostic method for N. gonorrhoeae should be specific, simple, sensitive, rapid and inexpensive. Currently, phenotypic identification methods have been dominated by NAAT methods, which allow detection and identification of gonococcus directly in the clinical specimen. However, up today molecular methods do not allow full determination of drug susceptibility.

Isolation and Identification of Biofilm forming Bacteria from Glass Panels exposed in Sea Waters of South Andaman, India

Marine organisms attach to surfaces that are submerged in seawater in a process known as biofouling. These organisms can colonize on various surfaces such as ship hulls, pillars, jetties, undersea pipelines, oil rigs and buoys. Foulers can include bacteria, algae, barnacles, mussels and other invertebrates. The initial adhesion of fouling bacteria is the first stage of biofouling. The growth and attachment of marine fouling bacteria are influenced by various environmental factors including salinity, temperature and nutrient availability. The bacteria forming marine biofilm were isolated and identified from glass panels. Basic microbiological phenotypic identification methods and genotypic validation using 16s rRNA gene sequencing were used to identify the bacterial strains. The phylogenetic analysis of the 16s rRNA gene sequences confirmed 13 different species (Bacillus aquimaris (2), Vibrio hepatarius (1), Exiguobacterium profundum (3), Exiguobacterium aestuarii (2), Vibrio parahaemolyticus (2), Vibrio alginolyticus (3), Vibrio azureus (2), Vibrio fluvialis (2), Priestia flexa (1), Pseudomonas aeruginosa (2), Bacillus megaterium (2), Lysinibacillus macroides (2) and Bacillus subtilis (1)) of fouling bacteria belonging to six genera.

Microbiological and Molecular Study of Paranasal Sinus Infections of Children with Malignancy and Unknown Origin Fever in Markazi Province, Iran

BackgroundChildren with malignancies are vulnerable to various infections, including sinus infections. Sinusitis is primarily caused by bacterial infections, followed by fungal infections. Due to this issue, evaluating the occurrence, diversity, and antibiotic susceptibility patterns of bacterial species that cause paranasal sinus infections in children with malignancy and unexplained fever is important. ObjectiveInvestigate the bacterial species accountable for sinusitis in children with malignancy and unexplained fever, and determine their susceptibility to antibiotics. MethodsThe study involved collecting 90 sinus samples from children aged 5-15 years with malignancy in Arak city. The isolates were identified using a combination of phenotypic, biochemical, and molecular techniques, including specific PCR and 16S rRNA gene sequencing. drug susceptibility testing was performed following the CLSI 2021 guidelines. ResultsA total of 36 isolates (40%) were obtained, including 4 isolates of Nocardia (11.12%), 4 isolates of E. coli (11.12%), 3 isolates of K. pneumoniae (8.33%), 5 isolates of P. aeruginosa (13.88%), 3 isolates of A. baumannii (8.33%), 4 isolates of S. aureus (11.12%), 3 isolates of S. epidermidis (8.33%), 5 isolates of S. agalactiae (13.88%), 2 isolates of S. pneumoniae (5.55%), and 3 isolates of E. faecium (8.33%). The isolates showed the most sensitivity to imipenem and trimethoprim-sulfamethoxazole and the least sensitivity to erythromycin and tetracycline. ConclusionsThe findings of the study indicate that sinusitis can contribute to fever of unknown origin in cancer patients. Therefore, it is recommended to use a combination of molecular and phenotypic methods for accurate identification of isolates. This approach can provide more reliable and precise results, leading to better diagnosis and treatment of sinusitis infections in children with malignancy.

Identification of Invasive Filamentous Mold Isolates Using DNA Sequencing: Experience of a Clinical Laboratory in a Resource-limited Setting

Background: Correct identification of clinically significant mold is becoming critical and cannot be relied only on phenotypic methods; hence, there is a dire need to develop an algorithm of workflows and capacity for molecular identification. This study shares the experience of DNA sequencing for invasive mold identification isolated and compared with phenotypic identification. Methods: This study was conducted at the microbiology laboratory, Aga Khan University, Karachi, Pakistan, and the Mycotic Disease Branch, Centers for Disease Control and Prevention, USA. Filamentous molds isolated from clinical specimens during January 2012–April 2013 were initially identified through phenotypic characteristics. Pan-fungal polymerase chain reaction targeting the internal transcribed spacer region of the ribosomal cistron and the D1/D2 domains of the 28S ribosomal cistron was performed. Sequencer version was utilized to edit and align the DNA sequences, and then sequences were identified using BLAST. The correlation between phenotypic and molecular identification was evaluated. Results: Gene sequencing identified 50% of clinical isolates as one of the Aspergillus species, followed by Mucorales 29%, Fusarium species 17%, and Cladosporium spp. 4%. Overall, 50% of clinical isolates were identified correctly till the species level by conventional methods. Phenotypic correlation with genotype till genus was 42%, while two isolates were wrongly identified phenotypically. Excellent agreement (100%) till species level between phenotypic and molecular identification for Aspergillus, while Mucorales had an agreement of 71%. Conclusion: Traditional phenotypic identification methods for filamentous molds had a good correlation with sequencing up to genus level identification; however, were not reliable up to species level.

Phenotypic and Genetic Characterization of Polyhydroxyalkanoate Producing Bacterial Isolates from Hypersaline Water Body, Atlantic Ocean, Nigeria

Introduction: The importance of polyhydroxylalkanoate (PHA) producing bacteria is hinged on the basis that PHA itself possess environmentally advantages over synthetic plastics therefore, the proper identity of these bacteria is inevitable.
 Aim: To determine the bacteria identification methods of phenotypic and genetic identification for marine sourced effective polyhydroxylalkanoate (PHA) production.
 Study Design: Randomised design was employed for PHA producing bacterial isolates.
 Place and Duration of Study: Samples containing PHA-producing bacteria from the hypersaline water body of Nigerian Southern Atlantic Ocean (6.35ᴼ N 3.28ᴼ E -St1; 6.35ᴼ N 3.40ᴼ E -St2; and 6.36ᴼ N 3.47ᴼ E -St3); water depths (0.07m (top), 50m (middle) and 100m (bottom) were collected and investigated between August, 2016-September, 2019.
 Methodology: Bacterial isolates was characterized using standard microbiological and biochemical tests for the phenotypes, and 16S rDNA for the genotypes. Isolates were also screened for PHA potentials using specific primers.
 Results: Genetically identified Bacillus cereus strains LB17 and Bacillus paramycoides SA27 were phenotypically identified as Bacillus badius. Similarly, Acinetobacter calcoaceticus JL11, five strains of Alcaligenes faecalis, seven strains of Bacillus spp., Enterobacter cloacae, Falsochrobactrum ovis, Ochrobactrum ciceri, Providencia stuartii including two species of Pseudomonas and Bordetella trematum were all phenotypically identified as Corynebacterium kutsceri. Invariably, Alcaligenes faecalis (strains PSD10 and DEP8), Pseudomonas aeruginosa (H47921) and Vagococcus fluvialis (AWW1) were identified as Staphylococcus species. All 34 bacterial isolates expressed phaC genes for PHA potential, while Alcaligenes faecalis strains and Bacillus humi (NBPP9) expressed phaC1 for short PHA chain.
 Conclusion: This study established variations in the genetical identity of Corynebacterium kutsceri, Enterobacter amnigenus, Micrococcus luteus, Micrococcus varians, Staphylococcus epidermidis and Staphylococcus saprophyticus when compared with the phenotypic identification methods. But, there was synchronization in the genotypic and phenotypic identity of bacillus species. Records of varietal differences of PHA potential were also obtained from same species in this research. It is therefore pertinent to rely on the genetic identification of bacteria for the effective determination of PHA-producing character as displayed in Bacillus spp.

Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan

BackgroundThe study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. MethodologyFrequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. ResultsWe found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. ConclusionBAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Discrepancies between phenotypic and genotypic identification methods of antibiotic resistant genes harboring Staphylococcusaureus

Antimicrobial resistance is a global issue that limits therapeutic options for infections. S. aureus being a member of the ESKAPE group is capable of “escaping” the biocidal action of antimicrobial agents. There are phenotypic and genotypic methods used for the identification of antibiotic resistant genes harboring S. aureus but these methods do not always show concordant results. To address these discrepancies, a total of 335 equine nasal swab samples from four districts of Punjab were collected using a convenient sampling technique. These samples were first subjected to common microbial techniques to identify S. aureus. The disc diffusion assay was performed for the phenotypic identification of antibiotic resistant S. aureus by using discs of oxacillin, penicillin, vancomycin, gentamycin, and tetracycline. After this, PCR was performed by targeting mecA, blaZ, vanB, aaca-aphd, and tetK genes for genotypic identification of respective antibiotic-resistant S. aureus. Phenotypic discrepancies (number of antibiotic resistant isolates found from disc diffusion who appeared to be negative for the resistant gene), and genotypic discrepancies (number of antibiotic sensitive isolates found from disc diffusion who appeared to be positive for the resistant gene) were calculated. The discrepancy ratio for mecA, blaZ, vanB, aaca-aphd, and tetK genes were 3.09, 1.96, 2.67, 1.93, and 1.67 respectively. These discrepant results indicate that the absence or presence of only one gene is not a true marker of resistant or sensitive isolates. There are multiple resistance determinants and resistance mechanisms. This study also highlighted the phenomenon of silencing of antibiotic resistance determinants.

Synthesis and Determination of Antimicrobial Efficacies of Secondary Metabolite of Streptomyces longisporoflavus and its 3D-Protein Structural Prediction

Aims: The need for new drug molecules is of high significance considering the rate at which pathogenic bacteria evolve into drug-resistant pathogens. Actinomycetes have been reported as valuable biological agents that possess potent bioactive molecules. This work aims to isolate local strains of actinomycetes in the environment and determine their antimicrobial activities against some clinical isolates.&#x0D; Study Design: This was an in-vitro study.&#x0D; Place and Duration of Study: The research was carried out at the Microbial Resources Research Laboratory, Department of Pure and Applied Biology, Ladoke Akintola University of Technology Ogbomoso, Nigeria between January 2020 and December, 2021.&#x0D; Methodology: Phenotypic and molecular methods of identification of the isolated microorganisms were done. A secondary metabolite of the isolate was obtained. An antibiotic sensitivity test of its metabolites was performed using ten (10) clinical isolates; inhibition zones were measured and recorded for each test organism. Gas chromatography-mass spectrometry (GC-MS) was used to determine the probable bioactive molecules present in the metabolite. The nucleotide sequences of the isolate were translated using Phyre2 and viewed with PyMOL.&#x0D; Results: The isolate was identified as Streptomyces longisporoflavus. Various zones of inhibition were recorded for each of the tested pathogenic organisms. Five (5) bioactive molecules were identified in the metabolites, with butane-1,1-dibutoxy-2 (1H)-quinolone having the highest peak area. The PYMOL result shows that the protein structure has a 64% identity as a binding molecule.&#x0D; Conclusion: This study reveals that the local isolates of S. longisporoflavus showed promising antimicrobial potential with bioactive molecules that are potent inhibitors of pathogenic organisms.

Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) as an Indispensable Tool in Diagnostic Bacteriology: A Comparative Analysis With Conventional Technique.

Owing to its accurate diagnosis, rapid turnaround time, cost effectivity, and less rates of error, Matrix-assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) has replaced most of the phenotypic methods of identification. Thus, the objective of this study was to compare and evaluate MALDI-TOF MS to conventional biochemical-to identify bacterial microorganisms. Different bacterial species isolated from 2010 to 2018 (pre-MALDI-TOF era), using routine bio-chemicals were compared to bacterial species isolated from 2019 to August 2021 (post MALDI-TOF), using MALDI-TOF, in the microbiology laboratory of a tertiary care hospital in North India. Chi-Square test (χ2) was used for the evaluation of bacterial identification between biochemical tests and MALDI-TOF MS association with a 95% confidence interval, considering wrong identification in genera or at a species level. Many different and new genera and species of bacteria could be identified using MALDI-TOF, which was not possible using only routine manual bio-chemicals like Kocuria rhizophilus, Rothia mucilaginosa,Enterococcus casseliflavus, Enterococcus gallinarum, Leuconostoc, Leclercia adecarboxylata, Raoultella ornithological, Cryseobacterium indologenes. Conclusion: Each of the newly identified bacteria played an important role in deciding treatment. Wide use of the MALDI-TOF system will not only strengthen diagnostic stewardship but also encourage antimicrobial stewardship programs.

Microbiological Characterization of Actinotignum schaalii Strains Causing Invasive Infections during a Multiyear Period in a Large Canadian Health Care Region.

Actinotignum schaalii is an underrecognized Gram-positive bacillus that is associated with urinary tract infections and cutaneous abscesses. The role of A. schaalii in invasive infections continues to be unappreciated because the bacteria can be isolated from a diverse spectrum of clinical specimens, ranging from being a single pathogen in urine and blood cultures to being deemed a colonizer in polymicrobial anaerobic cultures of sterile fluids and tissues. We conducted a microbiological analysis of clinical isolates obtained from 2012 through 2019. A total of 86 isolates were analyzed; 37 (43%) were from blood cultures, 35 (41%) were from deep wounds and abscesses, 6 (7%) were from urine samples, and the rest were recovered from peritoneal, kidney, and scrotal fluid samples. Urinary tract infections were clinically identified as the source of most cases of bacteremia, although no simultaneous urine cultures yielded positive results. The 16S rRNA gene sequences were available for 32 isolates (37%). Phylogenetic analysis revealed that AS.1/AS.2 strains caused a larger proportion of bloodstream infections (BSIs) (100% versus 52% [P = 0.01]) and trended toward a higher rate of hospitalization (91% versus 76% [P = 0.18]) but had a lower clindamycin MIC90 (0.12 versus >256 μg/mL). Our study emphasizes the emergence of A. schaalii as a pathogen in human urine samples, BSIs, and skin and soft tissue infections. It highlights the pitfalls of current laboratory methods in recovering and identifying this organism from clinical specimens, particularly urine samples. Phylogenetic analysis showed unique genotypic sequences for A. schaalii AS.1/AS.2 strains causing urosepsis, which requires further study to identify potential virulence factors. IMPORTANCE Actinotignum schaalii is an underrecognized Gram-positive bacillus due to its special growth requirements and prior phenotypic identification methods, and it is often mistaken as a contaminant. It has been associated with various clinical syndromes, from urinary tract infections to cutaneous infections. The widespread use of molecular diagnostic methods allowed for improved detection. However, its role in invasive infections remains underappreciated. We conducted a detailed microbiological analysis to improve our understanding of this organism's genotypic and phenotypic characteristics. Our results highlight the pitfalls of clinical laboratory recovery, particularly from urine cultures. Although most BSIs were caused by urinary tract infections, no simultaneous urine cultures identified A. schaalii, largely due to the failure of phenotypic methods to reliably isolate and identify this organism. Additionally, this is the first study demonstrating A. schaalii strains with differences in clinical and microbiological characteristics, raising the possibility of potential bacterial virulence factors contributing to invasive infections.

Cascade filtration and droplet digital detection integrated microfluidic assay enables isolating culture-free phenotypic identification of carbapenem-resistant organisms

Carbapenem-resistant organisms (CROs) are characterized by high drug resistance, rapid transmission, and high lethality. Therefore, rapid detection for CROs is essential for appropriate applying antibiotics and implementing quarantine. Droplet digital chromogenic assays (DDCA) have been accepted as an effective means for rapid microbial detection as the small droplet volumes facilitate a significant enhancement in the local concentration of chromogenic factors and, therefore, reduce the required time of the test. Nevertheless, as their dependence on the time-consuming isolation culture, the DDCA is still associated with a long turnaround time. To overcome this limitation, we develop here a microfluidic chip-based CRO phenotypic identification method that integrates cascade filtration (CF) with DDCA (CF-DDCA). After a body fluid sample is introduced to the microfluidic chip, particles with sizes >5 μm are removed out by the primary filter, and Gram (+) cocci with sizes <1 μm removed out by the secondary filter so that only Gram (−) bacilli with sizes between 1.5 and 5 μm are selectively retained. The purified Gram (−) bacilli, along with chromogenic reagents and carbapenem antibiotics, are then subjected to the DDCA. We demonstrate that the CF can remove 99.9% of the interfering microorganisms and thus eliminates the isolating culture. Benefited from the isolating culture-free DDCA, phenotypic identification of CROs can be achieved within 3.5 h. Clinical urine sample testing shows that the sensitivity and specificity of the CF-DDCA for CRO identification are all 100%, and the total coincidence rate between CF-DDCA and the conventional assay is also 100%.

​Application of MALDI-TOF Mass Spectrometry to Identify Lactic Acid Bacteria Isolated from Artisanal Dairy Products in Algeria

Background: Artisanal dairy products are considered an affordable food source and are highly valued by Algerian consumers. Methods: In this study during the period from 2019 to 2020, phenotypic identification methods and protein analysis by MALDI-TOF MS were performed to identify lactic acid bacteria isolated from 19 sample and five types of artisanal dairy products collected in different Algerian regions (East, West, North, South). Results: A number of 250 strains were isolated, only those that showed the characteristics of lactic acid bacteria were identified by analysing their proteins, The results showed that the strains belong to different species of LAB namely: Lactobacillus fermentum (31.03%), Enterococcus faecium (24.14%), Lactobacillus plantarum (21.55%), Enterococcus faecalis (17.24%), Lactococcus lactis ssp lactis (4.31%), Lactobacillus paracasei and Lactobacillus paraplantarum (0.86%). Identification by MALDI-TOF MS was similar to that provided by phenotypic Characterization. This new technique that has been used is considered as an effective tool for rapid identification of food-associated lactic acid strains and has also been successfully extended to dairy isolates.

P386 Molecular identification and antifungal susceptibility of fungi causing sinusitis in arid climate region

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesFungal sinusitis is a common problem worldwide with an increasing burden in arid climate regions. Clinical presentations of the disease range from allergic to acute invasive or chronic forms. In the present study, we aim to identify the etiology of fungal sinusitis in an arid climate region of Africa using molecular methods and to determine the antifungal susceptibility of the clinical isolates.MethodsA total of 60 isolates collected from patients diagnosed with fungal sinusitis in Khartoum, Sudan were included. Direct microscopic examination and culture from nasal biopsies were performed at the mycology reference laboratory in Khartoum. To confirm the presence of fungal elements in tissue, histology was performed for all samples. Cultured isolates were then identified by sequencing the internal transcribed spacer (ITS), β-tubulin, and calmodulin gene regions. Antifungal susceptibility was tested using the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST).ResultsSequencing of the three genes identified the following species: Aspergillus flavus (n = 48), A. citrinoterreus (n = 3), A. caespitosus (n = 2), A. sydowii (n = 2), Exserohilum sp. (n = 2), Purpureocillium lilacinum (n = 1), Trichosporon asahii (n = 1), and Fusarium chlamydosporum (n = 1).ConclusionThe fungi associated with sinusitis are of high diversity; the 60 isolates were found to belong to 8 different species. With the exception of cryptic species, a large concordance was found between molecular and phenotypic identification methods. Aspergillus is the most commonly reported genus with A. flavus as the most prevalent species.

P127 Cytokine gene Polymorphism in superficial Malassezia associated skin diseases

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectivesü To isolate and characterize Malassezia species from patients of Pityriasis Versicolor (PV), Atopic Dermatitis (AD), Seborrhoeic Dermatitis (SD), and healthy controls.ü To study single nucleotide polymorphism in IL-10 and IFN- γ genes of the host and its relation with susceptibility to Malassezia infection.MethodsIt was a prospective observational study done in University College of Medical Sciences and GTB Hospital, Delhi. Sample size comprised of 38 cases each of AD. Skin scrapings were used for fungal culture on Sabouraud Dextrose Agar (SDA) and Modified Dixon Agar (MDA) and isolates were identified as per conventional phenotypic methods. Genomic DNA was extracted from blood samples and Cytokine genotyping was carried out by Amplification Refractory Mutations System-Polymerase Chain Reaction (ARMS-PCR) with sequence-specific primers. Three SNPs (IL10-1082A/G; IL10-819/592C/T; IFN-g +874A/T) in two cytokine genes were assessed in all the patients and healthy controls. Chi-squared Test or Fisher's-Exact Test and Bonferroni's correction were used for statistical analysisResults Malassezia yeast was isolated in 94.7%, 63.1% each and 52.6% in PV, AD, SD, and healthy controls respectively. Malassezia globosa was the most commonly isolated species from both patient and healthy control. Malassezia sympodialis was the second most common followed by M. furfur and M. restricta. Association between specific cytokine gene polymorphism and clinical outcome was found to be significant in PV, AD, and SD group. IFN-γ +874 T allele and IFN-γ +874 A allele were significantly associated with PV and AD respectively. IFN-γ +874 AA genotype frequency was found to be higher in PV and AD patients than in controls. This finding suggests that PV and AD patients may produce a lower IFN-γ. IL10-819/592 C/T alleles were also significantly associated with PV. IL10-819/592 CT genotype frequency was found to be lower, and CC genotype frequency was found to be higher in PV patients as compared to healthy controls, suggesting that IL10 production may be higher in PV patients. AD patients were more likely to carry the IL10–1082 G allele and, it was significantly associated with this disease. Moreover, IL10-1082 AG genotype was significantly associated with AD and SD, which corresponds to high production of IL10.ConclusionThe identification of Malassezia yeast to a species level is of a great importance to determine which species are implicated in certain skin diseases.The use of phenotypic methods for identification of Malassezia species is a reliable, easily executed method, that is also inexpensive. Molecular methods are necessary to decrease the turnaround time, especially for slow-growing Malassezia species.Cytokine gene polymorphism studies in IL10 and IFN γ genes demonstrated susceptibility of host to Malassezia infections. Comparison with the serum cytokine levels will help in understanding the evolution of Malassezia infections in susceptible host.Population genetics studies require inclusion of a larger number of subjects to evaluate the probability or the frequency of occurrence of the genotype.

IDENTIFICATION PROFILE OF PREDOMINANT FUNGAL CONTAMINANTS OF DRY SEASON PRE-HARVEST GARDEN EGG CROP FROM UZO-UWANI, NSUKKA, NIGERIA FOR POSSIBLE CONTROL

In Nigeria, dry season garden eggs are often associated with higher fungal contamination than rain-fed garden eggs and most reports on the identified pathogens were on post-harvest crops. There is possibility that some of the implicated pathogens can be traced to the farm. Thus, the present study aimed to identify the predominant fungi contaminants of dry season pre-harvest garden egg for possible bio-control measures. Sampled pre-harvest garden eggs grouped into symptomatic and asymptomatic types were collected from three farms within January to March for two consecutive years. Standard phenotypic identification methods were adopted. The most predominant isolates were further subjected to ITS rRNA sequencing. The applied methods identified 7 common isolates across the farms in the ascending frequency of Fusarium thapsinum (6.67±10.34%) &lt;Colletotrichum spp (11.10±17.20%)&lt;Fusarium oxysporum (14.43±22.36%) &lt;Fusarium solani (23.70±20.32%) &lt;Penicillium citrinum (35.57±9.12%) &lt;Pichia spp (52.20±7.52%)&lt; Aspergillius niger (66.67±5.99%) which were all significant (P&lt;0.05). Among the isolates, a higher variance of 44.5 was observed within the three farms than within their months of recovery (14.8 variance). The fresh asymptomatic garden egg had 71.4% contaminants as against 100% from symptomatic ones. Molecular assay confirmed the most predominant isolates as Aspergillus niger 41 and Pichia kudriavzeviiC101 with assigned NCBI accession numbers of ON417062 and ON417063 after their sequence deposition in Genbank. High percentage recovery of these isolates especially from asymptomatic garden eggs gave an insight of possible health problems associated with it on consumption and this calls for appropriate bio-control measures from the farm

What's in a name? The importance of identity in heirloom apple tree preservation

Societal Impact StatementHistoric North American apple (Malus domestica) orchards that thrived in the late 19th and early 20th centuries, with cultivar compositions unlike today's orchards, are vanishing. There are several reasons for this loss: tree aging, cost of tree maintenance, and urbanization. Many groups have collected local knowledge regarding the history and horticulture of apples using both phenotypic and genotypic identification methods. Some of these groups have joined with scientists to form the collaborative “Historic Fruit Tree Working Group of North America” to facilitate the conservation of heirloom apple cultivars in North America through documentation, identification, collaboration, and education.

Coagulase-negative staphylococci as an evolving mastitis causing organism in cows: A review

The present review attempts to address the emergence of a fast-growing mastitis causing pathogen – coagulase-negative staphylococci (CNS) – and its multi-facet roles in dairy farms. CNS is divided into numerous species, but only a few succeed in bovine mastitis. The two predominant CNS species causing mastitis are Staphylococcus chromogenes and Staphylococcus simulans. These two mostly known CNS affects dairy cows at varied parities and at different lactation stages. CNS have been judged to be the most common bovine mastitis isolate in several countries of the world. However, CNS may not likely be as pathogenic as the other primary mastitis pathogens such as Staphylococcus aureus and its infection mostly remains subclinical. Conversely, CNS is capable of triggering persistent infections among dairy herds, resulting in reduced milk quality and high milk somatic cell count (SCC). One negative impact of CNS infection in dairy herds is that it can destroy cow udder tissue, hereby leading to a drop in milk production of dairy cows. CNS mastitis has a high cure rate when treated with anti-biotics as their infection in cows are not known to be a therapeutic problem. Judging from several literatures, it cannot be said whether CNS species act as an ecological or contagious pathogens. Regulator procedures, including pre- and post-milking teat disinfection, may reduce contagious mastitis pathogens of CNS infections in the dairy herd. Phenotypic methods for identification of CNS are not adequately dependable, and molecular techniques of identification may be better substitute for identification. Dairy farmers may benefit greatly from more empirical research on the epidemiology of CNS mastitis and more reliable methods for species identification.

Challenges in Serologic Diagnostics of Neglected Human Systemic Mycoses: An Overview on Characterization of New Targets.

Systemic mycoses have been viewed as neglected diseases and they are responsible for deaths and disabilities around the world. Rapid, low-cost, simple, highly-specific and sensitive diagnostic tests are critical components of patient care, disease control and active surveillance. However, the diagnosis of fungal infections represents a great challenge because of the decline in the expertise needed for identifying fungi, and a reduced number of instruments and assays specific to fungal identification. Unfortunately, time of diagnosis is one of the most important risk factors for mortality rates from many of the systemic mycoses. In addition, phenotypic and biochemical identification methods are often time-consuming, which has created an increasing demand for new methods of fungal identification. In this review, we discuss the current context of the diagnosis of the main systemic mycoses and propose alternative approaches for the identification of new targets for fungal pathogens, which can help in the development of new diagnostic tests.

Bacillus cereus Invasive Infections in Preterm Neonates: an Up-to-Date Review of the Literature.

Bacillus cereus group species are widespread, Gram-positive, spore-forming environmental bacteria. B. cereus sensu stricto is one of the major causes of food poisoning worldwide. In high-risk individuals, such as preterm neonates, B. cereus infections can cause fatal infections. It is important to note that the phenotypic identification methods commonly used in clinical microbiology laboratories make no distinction between B. cereus sensu stricto and the other members of the group (Bacillus anthracis excluded). As a result, all the invasive infections attributed to B. cereus are not necessarily due to B. cereus sensu stricto but likely to other closely related species of the B. cereus group. Next-generation sequencing (NGS) should be used to characterize the whole genome of the strains belonging to the B. cereus group. This could confirm whether the strains involved in previously reported B. cereus invasive infections preferentially belong to formerly known or emerging individual species. Moreover, infections related to B. cereus group species have probably been overlooked, since their isolation in human bacteriological samples has for a long time been regarded as an environmental contaminant of the cultures. Recent studies have questioned the emergence or reemergence of B. cereus invasive infections in preterm infants. This review reports our current understanding of B. cereus infections in neonates, including taxonomical updates, microbiological characteristics, bacterial identification, clinical features, host-pathogen interactions, environmental sources of contamination, and antimicrobial resistance.