Experimental evolution and genome data analysis ofCandida albicansreveals cryptic bacteria in single yeast colonies

ABSTRACTAt least 25% of patients with positiveCandida albicansbloodstream infection also have one or more bacterial species associated with the infection. These polymicrobial infections are usually caused by coagulase-negative staphylococci, most commonlyStaphylococcus epidermidisand are associated with significantly worse clinical outcomes as compared to monomicrobial infections. Here we show bacteria are present inC. albicanscultures started from isolated single colony platting. These co-evolving bacteria can only be detected by the use of specific selective medium and/or long periods of incubation from 8 days up to 48 weeks (approximately 4,000 generations), used in experimental evolution methods. The detection of these co-evolving bacteria is highly dependent on the type of enzyme used for 16S rRNA gene amplification and is often missed in clinical laboratory analysis because of short incubation periods, media and temperatures, used in mycology clinical routine, that are unfavorable for bacterial growth. In this study, we identified bacteria in cultures of differentC. albicansisolates from long term, continuous growth by molecular analysis and microscopy. Also, we confirmed the presence of these co-evolving bacteria by identification ofS. epidermidisgenome segments in sequencing reads of theC. albicansreference strain SC5314 genome sequencing project raw data deposited in GenBank. This result rules out the possibility of laboratory specific contamination. Also, we show that the presence of associated bacteria correlates with antifungal resistance alterations observed in growth under hypoxia. Our findings show the intense interaction betweenC. albicansyeasts and bacteria and have direct implications in yeast clinical procedures, especially concerning patient treatment.

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Microbial Diversity in Milk of Women With Mastitis: Potential Role of Coagulase-Negative Staphylococci, Viridans Group Streptococci, and Corynebacteria

Journal of Human Lactation ◽

10.1177/0890334417692968 ◽

2017 ◽

Vol 33 (2) ◽

pp. 309-318 ◽

Cited By ~ 23

Author(s):

Pilar Mediano ◽

Leonides Fernández ◽

Esther Jiménez ◽

Rebeca Arroyo ◽

Irene Espinosa-Martos ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Diversity ◽

Bacterial Species ◽

Total Bacterial Count ◽

Rrna Gene ◽

Coagulase Negative Staphylococci ◽

Milk Samples ◽

Lactational Mastitis ◽

Viridans Group Streptococci

Background: Lactational mastitis constitutes a significant cause of premature weaning. However, its etiology, linked to the presence of pathogenic microorganisms, has been scarcely reported. Research aim: The aim of this study was to describe the microbial diversity in milk samples from women suffering from lactational mastitis and to identify more accurately a collection of isolates belonging to coagulase-negative staphylococci, streptococci, and coryneform bacteria. Methods: This is a cross-sectional descriptive one-group study. A total of 5,009 isolates from 1,849 mastitis milk samples was identified by culture, biochemical, and/or molecular methods at the species or genus level. A more precise identification of a collection of 211 isolates was carried out by 16S rRNA gene sequencing. Results: Mean total bacterial count in milk samples was 4.11 log10 colony-forming units/ml, 95% confidence interval [4.08, 4.15]. Staphylococcus epidermidis was the most common species being isolated from 91.56% of the samples, whereas Staphylococcus aureus was detected in 29.74%. Streptococci and corynebacteria constituted the second (70.20%) and third (16.60%) most prevalent bacterial groups, respectively, found in this study. In contrast, Candida spp. was present in only 0.54% of the samples. Sequencing of the 16S rRNA gene revealed a high diversity of bacterial species among identified isolates. Conclusion: Many coagulase-negative staphylococci, viridans group streptococci, and corynebacteria, usually dismissed as contaminant bacteria, may play an important role as etiologic agents of mastitis. Proper diagnosis of mastitis should be established after performing microbiological testing of milk based on standardized procedures. A reliable analysis must identify the mastitis-causing pathogen(s) at the species level and its(their) concentration(s).

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DETEKSI FENOTIPIK Methicillin Resistant Staphylococcus aureus (MRSA) PADA SAMPEL MAKANAN DI SIDOARJO

Meditory : The Journal of Medical Laboratory ◽

10.33992/m.v7i1.554 ◽

2019 ◽

Vol 7 (1) ◽

pp. 55-65

Author(s):

Yulianto Ade Prasetya

Keyword(s):

Clinical Laboratory ◽

Bacterial Species ◽

Antibiotic Sensitivity ◽

Food Samples ◽

Selective Medium ◽

Beta Lactam ◽

Biochemical Tests ◽

Methicillin Resistant ◽

Staphyloccocus Aureus ◽

Fried Foods

Methicillin-resistant Staphyloccocus aureus (MRSA) is an isolate that is resistant to the antibiotic methicillin and beta lactam group. The incidence of MRSA associated with nosocomial infections in various parts of the world is very high, but research on its spread in community infections is rarely reported. This study aims to detect the presence of phenotypic MRSA in food samples in Sidoarjo. The food samples (cilok, fried foods and tempura) collected were then weighed, diluted, and cultured in a selective medium and differential namely Manitol Salt agar. The yellow-colored colonies were then continued with microscopic testing and biochemical tests to distinguish between Staphylococcus species. Thirty eight collected Staphyloccus aureus isolates were then screened using Oxacillin 1 µg and there were eight (8) isolates that were positive for MRSA according to the criteria of the Clinical Laboratory Standart Institutre (CLSI). Eight of the isolates were tested for antibiotic sensitivity with the Kirby-Bauer method with Chloramphenicol 30 µg and Cotrimoxazole 25 µg. Eight MRSA (21%) isolates were resistant to Chloramphenicol and only four isolates were resistant to Cotrimoxazole. The presence of MRSA isolates in community infections needs to be watched out for considering these genes can be transmitted and spread between bacterial species

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Antibiotic Resistance of Coagulase-Negative Staphylococci and Lactic Acid Bacteria Isolated from Naturally Fermented Chinese Cured Beef

Journal of Food Protection ◽

10.4315/0362-028x.jfp-18-195 ◽

2018 ◽

Vol 81 (12) ◽

pp. 2054-2063 ◽

Cited By ~ 1

Author(s):

JING WANG ◽

MINGYUE LI ◽

JING WANG ◽

MIAOMIAO LIU ◽

KUN YANG ◽

...

Keyword(s):

Antibiotic Resistance ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Random Amplified Polymorphic Dna ◽

Selective Medium ◽

Dilution Method ◽

Agar Dilution Method ◽

Rrna Gene ◽

M Gene ◽

Coagulase Negative Staphylococci

ABSTRACT This study provided phenotypic and molecular analysis of the antibiotic resistance within coagulase-negative staphylococci and lactic acid bacteria isolated from naturally fermented Chinese cured beef. A total of 49 strains were isolated by selective medium and identified at the species level by 16S rRNA gene sequencing as follows: Staphylococcus carnosus (37), Lactobacillus plantarum (6), Weissella confusa (4), Lactobacillus sakei (1), and Weissella cibaria (1). All strains were typed by random amplified polymorphic DNA fingerprinting, and their antibiotic resistances profiles to 15 antibiotics were determined as the MIC by using the agar dilution method. All the tested strains were sensitive to ampicillin, and most of them were also sensitive to penicillin, gentamycin, neomycin, norfloxacin, and ciprofloxacin with low MICs. High resistance to streptomycin, vancomycin, erythromycin, roxithromycin, lincomycin, and kanamycin was widely observed, while the resistant levels to tetracycline, oxytetracycline, and chloramphenicol varied. The presence of corresponding resistance genes in resistant isolates was investigated by PCR, with the following genes detected: tet(M) gene in 9 S. carnosus strains and 1 W. confusa strain; erm(F) gene in 10 S. carnosus strains; ere(A) gene in 6 S. carnosus strains; ere(A) gene in 4 S. carnosus strains and 1 L. plantarum strain; and str(A) gene and str(B) gene in 3 S. carnosus strains. The results indicated that multiple antibiotic resistances were common in coagulase-negative staphylococci and lactic acid bacteria strains isolated from naturally fermented Chinese cured beef. Safety analysis and risk assessment should be performed for application in meat products.

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A PCR Detection Method for Rapid Identification of Paenibacillus larvae

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.2243-2245.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 2243-2245 ◽

Cited By ~ 46

Author(s):

V. A. Govan ◽

M. H. Allsopp ◽

S. Davison

Keyword(s):

Detection Method ◽

Bacterial Species ◽

Pcr Primers ◽

Rapid Identification ◽

Selective Medium ◽

Pcr Detection ◽

Paenibacillus Larvae ◽

Rrna Gene ◽

American Foulbrood ◽

The 16S Rrna Gene

ABSTRACT American foulbrood is a disease of larval honeybees (Apis mellifera) caused by the bacterium Paenibacillus larvae. Over the years attempts have been made to develop a selective medium for the detection of P. larvae spores from honey samples. The most successful of these is a semiselective medium containing nalidixic acid and pipermedic acid. Although this medium allows the growth of P. larvae and prevents the growth of most other bacterial species, the false-positive colonies that grow on it prevent the rapid confirmation of the presence ofP. larvae. Here we describe a PCR detection method which can be used on the colonies that grow on this semiselective medium and thereby allows the rapid confirmation of the presence ofP. larvae. The PCR primers were designed on the basis of the 16S rRNA gene of P. larvae and selectively amplify a 973-bp amplicon. The PCR amplicon was confirmed as originating from P. larvae by sequencing in both directions. Detection was specific for P. larvae, and the primers did not hybridize with DNA from closely related bacterial species.

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DIVERSITY OF CULTURABLE BACTERIAL MICROBIOTA OF THE Eisenia foetida DIGESTIVE TRACT

Revista Fitotecnia Mexicana ◽

10.35196/rfm.2018.3.255-264 ◽

2018 ◽

Vol 41 (3) ◽

pp. 255-264 ◽

Cited By ~ 1

Author(s):

J. Abraham Pérez-Pérez ◽

David Espinosa-Victoria ◽

Hilda V. Silva-Rojas ◽

Lucía López-Reyes

Keyword(s):

Bacterial Diversity ◽

Digestive Tract ◽

Bacterial Species ◽

Brain Heart Infusion ◽

Rrna Gene ◽

Eisenia Foetida ◽

Bacterial Isolates ◽

Bacterial Microbiota ◽

Decomposition Of Organic Matter ◽

Earthworm Gut

Bacteria are an unavoidable component of the natural earthworm diet; thus, bacterial diversity in the earthworm gut is directly linked to decomposition of organic matter and development of the surrounding plants. The aim of this research was to isolate and to identify biochemically and molecularly the culturable bacterial microbiota of the digestive tract of Eisenia foetida. Earthworms were sourced from Instituto de Reconversión Productiva y Bioenergética (IRBIO) and Colegio de Postgraduados (COLPOS), México. Bacterial isolation was carried out on plates of Brain Heart Infusion (BHI) culture medium. Fifty six and 44 bacterial isolates were obtained from IRBIO and COLPOS, respectively. The population was composed of 44 Gram-negative and 56 Gram-positive isolates. Over 50 % of the bacterial isolates were rod-shaped cells. The 16S rRNA gene was sequenced and nine genera were identified in worms from IRBIO (Bacillus, Paenibacillus, Solibacillus, Staphylococcus, Arthrobacter, Pantoea, Stenotrophomonas, Acinetobacter and Aeromonas) and six in worms from COLPOS (Bacillus, Paenibacillus, Stenotrophomonas, Staphylococcus, Acinetobacter and Aeromonas). Bacillus was the predominant genus, with eight and six species in the oligochaetes from IRBIO and COLPOS, respectively. The most represented bacteria in the worms from both sites were Bacillus sp. and B. subtilis. The predominance of Bacillus was probably due to spore formation, a reproductive strategy that ensures survival and dispersion in the soil and oligochaetes digestive tract. The gut of E. foetida not only harbored bacterial species of agronomic importance but also species potentially pathogenic for humans (Staphylococcus warneri, Pantoea agglomerans and Stentrophomonas sp.). The larger bacterial diversity in worms from IRBIO could be due to their feeding on cattle manure, which is a rich source of bacteria.

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Clinical Implications of Polymicrobial Synergism Effects on Antimicrobial Susceptibility

Pathogens ◽

10.3390/pathogens10020144 ◽

2021 ◽

Vol 10 (2) ◽

pp. 144

Author(s):

William Little ◽

Caroline Black ◽

Allie Clinton Smith

Keyword(s):

Antimicrobial Susceptibility ◽

Chronic Wounds ◽

Clinical Laboratory ◽

Patient Treatment ◽

Clinical Implications ◽

Clinical Environment ◽

Tolerance Mechanisms ◽

Sequencing Technologies ◽

Generation Sequencing ◽

Polymicrobial Infections

With the development of next generation sequencing technologies in recent years, it has been demonstrated that many human infectious processes, including chronic wounds, cystic fibrosis, and otitis media, are associated with a polymicrobial burden. Research has also demonstrated that polymicrobial infections tend to be associated with treatment failure and worse patient prognoses. Despite the importance of the polymicrobial nature of many infection states, the current clinical standard for determining antimicrobial susceptibility in the clinical laboratory is exclusively performed on unimicrobial suspensions. There is a growing body of research demonstrating that microorganisms in a polymicrobial environment can synergize their activities associated with a variety of outcomes, including changes to their antimicrobial susceptibility through both resistance and tolerance mechanisms. This review highlights the current body of work describing polymicrobial synergism, both inter- and intra-kingdom, impacting antimicrobial susceptibility. Given the importance of polymicrobial synergism in the clinical environment, a new system of determining antimicrobial susceptibility from polymicrobial infections may significantly impact patient treatment and outcomes.

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Nanopore-Sequencing Characterization of the Gut Microbiota of Melolontha melolontha Larvae: Contribution to Protection against Entomopathogenic Nematodes?

Pathogens ◽

10.3390/pathogens10040396 ◽

2021 ◽

Vol 10 (4) ◽

pp. 396

Author(s):

Ewa Sajnaga ◽

Marcin Skowronek ◽

Agnieszka Kalwasińska ◽

Waldemar Kazimierczak ◽

Karolina Ferenc ◽

...

Keyword(s):

Gut Microbiota ◽

Entomopathogenic Nematodes ◽

Bacterial Species ◽

Antagonistic Activity ◽

Rrna Gene ◽

Nanopore Sequencing ◽

Bacterial Phyla ◽

Melolontha Melolontha

This study focused on the potential relationships between midgut microbiota of the common cockchafer Melolontha melolontha larvae and their resistance to entomopathogenic nematodes (EPN) infection. We investigated the bacterial community associated with control and unsusceptible EPN-exposed insects through nanopore sequencing of the 16S rRNA gene. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes were the most abundant bacterial phyla within the complex and variable midgut microbiota of the wild M. melolontha larvae. The core microbiota was found to include 82 genera, which accounted for 3.4% of the total number of identified genera. The EPN-resistant larvae differed significantly from the control ones in the abundance of many genera belonging to the Actinomycetales, Rhizobiales, and Clostridiales orders. Additionally, the analysis of the microbiome networks revealed different sets of keystone midgut bacterial genera between these two groups of insects, indicating differences in the mutual interactions between bacteria. Finally, we detected Xenorhabdus and Photorhabdus as gut residents and various bacterial species exhibiting antagonistic activity against these entomopathogens. This study paves the way to further research aimed at unravelling the role of the host gut microbiota on the output of EPN infection, which may contribute to enhancement of the efficiency of nematodes used in eco-friendly pest management.

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A Combined Analysis of Gut and Skin Microbiota in Infants with Food Allergy and Atopic Dermatitis: A Pilot Study

Nutrients ◽

10.3390/nu13051682 ◽

2021 ◽

Vol 13 (5) ◽

pp. 1682

Author(s):

Ewa Łoś-Rycharska ◽

Marcin Gołębiewski ◽

Marcin Sikora ◽

Tomasz Grzybowski ◽

Marta Gorzkiewicz ◽

...

Keyword(s):

Atopic Dermatitis ◽

Food Allergy ◽

Gut Microbiota ◽

Bacterial Species ◽

Rrna Gene ◽

Healthy Children ◽

Skin Microbiota ◽

Fecal Samples ◽

Healthy Infants ◽

Α Diversity

The gut microbiota in patients with food allergy, and the skin microbiota in atopic dermatitis patients differ from those of healthy people. We hypothesize that relationships may exist between gut and skin microbiota in patients with allergies. The aim of this study was to determine the possible relationship between gut and skin microbiota in patients with allergies, hence simultaneous analysis of the two compartments of microbiota was performed in infants with and without allergic symptoms. Fifty-nine infants with food allergy and/or atopic dermatitis and 28 healthy children were enrolled in the study. The skin and gut microbiota were evaluated using 16S rRNA gene amplicon sequencing. No significant differences in the α-diversity of dermal or fecal microbiota were observed between allergic and non-allergic infants; however, a significant relationship was found between bacterial community structure and allergy phenotypes, especially in the fecal samples. Certain clinical conditions were associated with characteristic bacterial taxa in the skin and gut microbiota. Positive correlations were found between skin and fecal samples in the abundance of Gemella among allergic infants, and Lactobacillus and Bacteroides among healthy infants. Although infants with allergies and healthy infants demonstrate microbiota with similar α-diversity, some differences in β-diversity and bacterial species abundance can be seen, which may depend on the phenotype of the allergy. For some organisms, their abundance in skin and feces samples may be correlated, and these correlations might serve as indicators of the host’s allergic state.

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Evaluation of Commercial Disinfectants against Staphylococcus lentus and Micrococcus spp. of Poultry Origin

Veterinary Medicine International ◽

10.1155/2020/8811540 ◽

2020 ◽

Vol 2020 ◽

pp. 1-10

Author(s):

Otun Saha ◽

Nadira Naznin Rakhi ◽

Arif Istiaq ◽

Israt Islam ◽

Munawar Sultana ◽

...

Keyword(s):

Micrococcus Luteus ◽

Bacterial Species ◽

16S Rrna Gene Sequencing ◽

Average Diameter ◽

Diffusion Method ◽

Dilution Method ◽

Rrna Gene ◽

Poultry Farms ◽

Rrna Gene Sequencing ◽

Selection Of

Introduction. Effective sanitation strategies for poultry farms require an appropriate selection of the disinfectant based on the contaminants present and their sensitivity to the disinfectants. Aim. The current study investigated the prevalence of streptococci/micrococci in poultry farms of Bangladesh and the efficacy of commercial disinfectants (Savlon, Lysol, Quatovet, Virkon S, and Virocid) along with alcohol against these pathogens to adopt appropriate strategies. Materials and Methods. Conventional approaches and the 16S rRNA gene sequencing were performed to confirm the isolates at the species level along with microtiter biofilm assay to determine their biofilm-forming ability. Efficacy of the disinfectants was tested against those isolates using agar well diffusion and minimum inhibitory concentration (MIC) test by broth dilution method using different dilutions of the disinfectants. Results. Staphylococcus lentus (n = 32), Micrococcus luteus (n = 7), and Micrococcus aloeverae (n = 4) were confirmed among 102 presumptively screened streptococci/micrococci isolates from 43 samples. No single disinfectant showed equally high efficacy against all three bacterial species in agar well diffusion test, although Virocid showed the lowest MIC against all three of them. Lysol was least effective among the commercial disinfectants by both MIC and diffusion method, although each commercial disinfectant was more effective than alcohol. Considering both the average diameter of the inhibition zones and the MIC values, efficacy can be interpreted as Virocid > Quatovet > Savlon > Virkon S > Lysol. Although the efficacy decreased with decreasing concentration, the disinfectants retained a satisfactory level of efficacy at 50% concentration. Among test pathogens, M. aloeverae was the most sensitive to the disinfectants and the weakest biofilm producers, whereas 4/14 S. lentus and 1/5 M. luteus were strong biofilm producers, which may cause more reduction in the efficacy in environmental conditions. Conclusion. As no ideal disinfectant was found in the study, the efficacy of the disinfectants should be routinely evaluated and validated to ensure the sanitation standards in the poultry sector.

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Detection of Food Spoilage and Pathogenic Bacteria Based on Ligation Detection Reaction Coupled to Flow-Through Hybridization on Membranes

BioMed Research International ◽

10.1155/2014/156323 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 7

Author(s):

K. Böhme ◽

P. Cremonesi ◽

M. Severgnini ◽

Tomás G. Villa ◽

I. C. Fernández-No ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Bacterial Species ◽

Cost Effective ◽

Rrna Gene ◽

Bacterial Identification ◽

Dot Blot ◽

Culture Collections ◽

Ligation Detection Reaction ◽

Flow Through ◽

Food Safety And Quality

Traditional culturing methods are still commonly applied for bacterial identification in the food control sector, despite being time and labor intensive. Microarray technologies represent an interesting alternative. However, they require higher costs and technical expertise, making them still inappropriate for microbial routine analysis. The present study describes the development of an efficient method for bacterial identification based on flow-through reverse dot-blot (FT-RDB) hybridization on membranes, coupled to the high specific ligation detection reaction (LDR). First, the methodology was optimized by testing different types of ligase enzymes, labeling, and membranes. Furthermore, specific oligonucleotide probes were designed based on the 16S rRNA gene, using the bioinformatic tool Oligonucleotide Retrieving for Molecular Applications (ORMA). Four probes were selected and synthesized, being specific forAeromonasspp.,Pseudomonasspp.,Shewanellaspp., andMorganella morganii, respectively. For the validation of the probes, 16 reference strains from type culture collections were tested by LDR and FT-RDB hybridization using universal arrays spotted onto membranes. In conclusion, the described methodology could be applied for the rapid, accurate, and cost-effective identification of bacterial species, exhibiting special relevance in food safety and quality.

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