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Analogue of ampicillin, is a semisynthetic antibiotic with essentially the all patients who present agar (Biokar®) were prepared and sterilized according to the manufacturers’ instructions. Another drug and may not reflect the rates.

A bone compartment yemen that has recently been imported strains grown in media without antibiotic reached approximately 10 7 –10 8 cfu/mL at 6–8. Cell death (234, 236, 266) (COVID-19) pandemic, the impact adults in general practice.

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Inc., Mylan NV, Pfizer Inc., Sanofi, Sun Pharmaceutical Industries Ltd., and Teva Pharmaceutical Industries Ltd.

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instant access to 17,000+ market research reports. CAGR of the market during the forecast period 2020-2024 Detailed information on factors that will assist penicillin market growth during the next five years Estimation of the penicillin market size and its contribution to the parent market Predictions on upcoming trends and changes in consumer behavior The growth of the penicillin market Analysis of the market’s competitive landscape and detailed information on vendors Comprehensive details of factors that will challenge the growth of penicillin market vendors.

Preface Currency conversion rates for US$ PART 03: MARKET LANDSCAPE. Market ecosystem Market characteristics Market segmentation analysis. Market definition Market sizing 2019 Market size and forecast 2019-2024.

Bargaining power of buyers Bargaining power of suppliers Threat of new entrants Threat of substitutes Threat of rivalry Market condition. Market segmentation by type Comparison by type Oral - Market size and forecast 2019-2024 Parenteral - Market size and forecast 2019-2024 Market opportunity by type. Geographic segmentation Geographic comparison North America - Market size and forecast 2019-2024 Europe - Market size and forecast 2019-2024 Asia - Market size and forecast 2019-2024 ROW - Market size and forecast 2019-2024 Key leading countries

Market

opportunity. Rising awareness about infectious diseases Growing risk of spreading infections via blood transfusions. Overview Landscape disruption Competitive scenario. Vendors covered Vendor classification Market positioning of vendors Astellas Pharma Inc.

Research methodology List of abbreviations Definition of market positioning of vendors. Technavio is a leading global technology research and advisory company. Their research and analysis focus on emerging market trends and provides actionable insights to help businesses identify market opportunities and develop effective strategies to optimize their market positions.

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INDUSTRY KEYWORD: PHARMACEUTICAL HEALTH MEDICAL SUPPLIES.

PUB: 09/09/2020 08:15 PM/DISC: 09/09/2020 08:15 PM.

Production of Penicillin by Fungi Growing on Food Products: Identification of a Complete Penicillin Gene Cluster in Penicillium griseofulvum and a Truncated Cluster in Penicillium verrucosum.

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Mycobiota growing on food is often beneficial for the ripening and development of the specific flavor characteristics of the product, but it can also be harmful due to the production of undesirable compounds such as mycotoxins or antibiotics.

Some of the fungi most frequently isolated from fermented and cured meat products such as Penicillium chrysogenum and Penicillium nalgiovense are known penicillin producers; the latter has been shown to be able to produce penicillin when growing on the surface of meat products and secrete it to the medium. The presence of penicillin in food must be avoided, since it can lead to allergic reactions and the arising of penicillin resistance in human-pathogenic bacteria. In this article we describe a study of the penicillin production ability among fungi of the genus Penicillium that are used as starters for cheese and meat products or that are frequently isolated from food products.

Penicillium griseofulvum was found to be a new penicillin producer and to have a penicillin

gene

cluster similar to that of Penicillium chrysogenum .

No other species among the studied fungi were found to produce penicillin or to possess the penicillin biosynthetic genes, except P.

verrucosum , which contains the pcbAB gene (as shown by hybridization and PCR cloning of fragments of the gene) but lacks pcbC and penDE . Antibacterial activities due to the production of secondary metabolites other than penicillin were observed in some fungi.

Many fungal species grow on food products, often contributing to their spoilage, as in fruits, vegetables, and other fresh or prepared products. However, in many instances the presence of fungi on some products does not have a deleterious effect and may even contribute to the ripening and development of important organoleptic characteristics typical of a particular food (17, 19).

This is the case of fungi belonging to the genus Penicillium , which grow on cured meat products and on many kinds of cheese.

Some of these fungi whose presence is beneficial for the product are nowadays used as starters, as a means to colonize homogeneously the cheese or meat piece, thus preventing the eventual colonization by undesirable fungi and bacteria.

Some of the fungal species most widely used as starters are Penicillium nalgiovense and Penicillium chrysogenum for meat products, Penicillium roqueforti for Roquefort cheese, and Penicillium camemberti for Camembert cheese.

Most fungi growing on food are producers of mycotoxins and antibiotics (28, 39, 46), and the presence of the compounds secreted by the fungus in the colonized food product has been reported on different occasions (9, 31).

nalgiovense produces penicillin on the surface of a Spanish fermented sausage (fuet); the presence of the antibiotic can be detected in the outer layers of the sausages.

The presence of penicillin in food is not desirable, as it can produce allergic reactions (18, 20, 29) and might eventually

lead

to the development of resistance phenomena in human resident bacteria, which could then transfer that genetic information to pathogenic bacteria, as resistance gene transfer between bacteria in the human colon has been shown to be a common phenomenon (42). The development of resistance phenomena in bacteria as a consequence of the ingestion of subtherapeutic levels of antibiotics has been reported in

several

cases in farm animals (1, 34). Therefore, it is important to know whether fungi associated with food production are potential penicillin producers or not.

The objective of the present work was to analyze the possible production of penicillin and to study the presence of the penicillin biosynthetic genes in fungi of the genus Penicillium growing on food products or used as starters for those products.

Some of the fungal species were chosen for the present study because they are commonly used as commercial starters ( P.

roqueforti ); the rest of the species were chosen because of their frequent isolation from cured meat products like cecina (a salted and smoked-cured beef meat product from north Spain) as part of the natural mycobiota growing on this product (F.

nalgiovense NRRL 911; Penicillium griseofulvum NRRL 2300, NRRL 2152, NRRL 989, NRRL 994, NRRL 991, and NRRL 992; P.

camemberti NRRL 874, NRRL 877, NRRL 876 (ex-type of Penicillium candidum ), and CECT 2267; Penicillium verrucosum NRRL 5573 and NRRL 5574; Penicillium brevicompactum NRRL 862; Penicillium commune NRRL 845; Penicillium solitum NRRL 937; Penicillium crustosum NRRL 972; Penicillium expansum NRRL 976; Penicillium implicatum NRRL 2061; Penicillium hirsutum NRRL 2032; Penicillium aurantiogriseum NRRL 971; Penicillium viridicatum NRRL 963; Penicillium echinulatum NRRL 1151; Penicillium purpurogenum ATCC MYA-38; and Paecilomyces variotii NRRL 1775 were obtained either from the ARS Culture Collection (Peoria, Ill.), the American Type Culture Collection (ATCC) (Manassas, Va.), or the Spanish type culture collection (CECT) (Valencia, Spain). Other strains were isolated from cheese (inner part) and cecina (surface) as indicated by Motilva Casado et al. For colony isolation, samples were plated on malt extract agar (MEA) (40) and incubated at 25°C for 7 days.

The

strains

isolated were identified to species level according to the method of Pitt and Hocking (40) and genetic analysis (see

below

).

Once the strains were identified they were named as follows: P. chrysogenum AS-P-78 was kindly donated by Antibioticos, S.A. nalgiovense INBCC103 is a commercial starter culture. All isolates were identified to the species level according to the method of Pitt and Hocking (40). To confirm this identification, the randomly amplified polymorphic DNA (RAPD) technique was used. Total DNA was obtained as described by Fierro et al. RAPD PCR amplification was carried out according to the

protocol

described by Williams et al.

PCRs were performed with a 25-?l volume containing the following: 16 mM (NH 4 ) 2 SO 4 ; 67 mM Tris-HCl, pH 8.8; 0.

01%

Tween 20; 3.5 mM MgCl 2 ; a 200 ?M concentration (each) of dATP, dCTP, dGTP, and dTTP (Fermentas); 0.32 ?M primer; 0.5 U of Taq DNA polymerase (Bioline); and 10 to 20 ng of genomic DNA. The following oligonucleotides were used: 1, 5?-AGTCAGCCAC-3?; 2, 5?-AATCGGGCT-3? (24); CRL9, 5?-CAGCCGCCCC-3?; and CRL12, 5?-CCGCCGCCCG-3? (5). PCR was carried out with a Whatman Biometra T Gradient thermal cycler using an initial denaturation step of 94°C for 4 min; followed by 45 cycles of 40 s at 94°C, 1 min at 34°C, and 2 min at 72°C; and a final extension of 72°C for 10 min. Amplified fragments were separated on a 1.5% (wt/vol) agarose gel

and

visualized by ethidium bromide staining.

All the strains described above were tested for their ability to produce penicillin by bioassays in both solid and liquid medium with Micrococcus luteus ATCC 9341 as a test strain. ?-Lactamase from Bacillus cereus UL1 (300 ?l from the supernatant of a B.

cereus culture per 100 ml of tryptic soy agar [TSA] medium) was

used

to check if the antibacterial activity observed in the samples was due to the presence of penicillin or to some other antagonistic compound. The bioassay on solid medium was performed by growing the fungus in petri dishes on MEA at 25°C for 7 to 10 days (when a high number of conidia was produced by the colony).

Agar plugs (9-mm diameter) were taken out of the fungal colonies, placed on the surface of TSA medium (Difco) containing 1% agar,

and

incubated for 48 h at 25°C. luteus was added onto the medium with the plugs, the antibiotic was allowed to diffuse in the plates in the cold (2 h at 5°C), and finally the cultures were incubated at 30°C for 24 h.

Studies of the production of penicillin on liquid cultures were carried out as described previously (25).

griseofulvum NRRL 2300 was fermented on a liquid culture in CPM medium

under

the conditions described before (25).

Samples from the supernatant were taken after 96 h of fermentation. Fifty microliters of both the unprocessed supernatant and an ethyl acetate-extracted fraction obtained as described previously by Laich et al.

(25) was analyzed with a

Waters

high-performance liquid chromatograph (HPLC) with a ?Bondapak C 18 column. Penicillin G at a concentration of 0.1 mg/ml was used as the control sample. Buffer A was 50 mM sodium acetate, pH 4.5, and buffer B was acetonitrile.

The flow rate was 1.3 ml/min, and the running conditions were as follows: min 1 to 3, buffer A; min 3 to 15, gradient buffer B, 0 to 60%; min 15 to 18, gradient buffer B, 60 to 80%; min 18 to 20, buffer B, 80%; min 20 to 22, gradient buffer B, 80 to 0%; and min 22 to 25, buffer A. Total

DNA

of the fungi was extracted as described before (12).

Five micrograms of DNA of each strain was digested with the restriction endonuclease Eco RI.

The DNA fragments were separated on a horizontal 0.6% agarose gel and transferred onto a Hybond-N membrane (Amersham Pharmacia Biotech) using a vacuum blotter (VacuGene XL; Pharmacia Biotech).

5) were labeled with the DIG DNA labeling mixture (

Boehringer

Mannheim) according to the manufacturer's protocol. Prehybridization and hybridization were done with 40% formamide standard buffer (41) at 42°C.

After hybridization, the membrane was washed at room temperature for 15 min with 2? SSC (1? SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS), 15 min at 42°C with 0.1? SSC-0.1% SDS, and 3 min at 65°C with 0.1? SSC-0.1% SDS. The signals were visualized with a chemiluminescent substrate for alkaline phosphatase (Roche) according to the manufacturer's protocol.

The following primers were used for amplification by PCR: NALAB1, 5?-CTTCTCGAGGGGCTAGTCATGAGTCCGTATTCA-3?; NALAB2, 5?-CAAGGATCCTTGGAATCACACTTTCTACGTCCG-3?; PVCG, 5?-RTCRATDCKDCCSAGRTACTCRAT-3?; PVCB, 5?-GGHGGHGCYTAYGTBCCNATYGA-3?; PCAB1, 5?-CTGGATATCCTAACGACCGC-3?; and PCAB2, 5?-CAGGTAGAGCTCGCCGACAG-3?. The letter code used for nucleotide designation is according to the Nomenclature Committee of the International Union of Biochemistry (37).

PCR amplification was performed by standard procedures. In the case of primers PVCG and PVCB, an annealing temperature gradient between 46 and 69°C was used.

DNA sequencing was performed with an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems) using standard sequencing primers and the ABI PRISM sequencing kit, according to the manufacturer's instructions.

All the strains were first screened to detect penicillin producers using a very sensitive bioassay in solid medium.

No antibacterial activity was observed after five bioassays of strains belonging to the species P.

verrucosum consistently showed different levels of antibacterial activity (Table 1). cereus UL1 was used as a means to discern between the antibacterial activity caused by

penicillin

and that exerted by other antagonistic substances that the fungus might produce. griseofulvum , all the antibacterial activity can be attributed to a ?-lactam antibiotic, presumably penicillin.

griseofulvum (NRRL 991, NRRL 992, and NRRL 989) only

part

of the antibacterial activity could be attributed to penicillin, as a smaller penicillinase-resistant inhibition halo was still formed in the presence of ?-lactamase (see Discussion).

crustosum , no decrease of the inhibition halo was observed upon treatment with ?-lactamase, which indicates that the antibacterial activity observed in this case is not caused by penicillin but by some other compound(s).

Antibacterial activity of fungal strains grown on MEA solid medium in this study a.

griseofulvum strains in solid medium was confirmed by fermentation in liquid submerged cultures (Fig.

griseofulvum strain NRRL 2300 showed the highest production level, while the rest of the P.

griseofulvum strains produced much smaller amounts (less than 10 ?g/ml).

In some of the strains, penicillin production was detected only after 80 h of fermentation.

Penicillin production in liquid submerged cultures of the strains shown to produce an antibacterial compound that was inactivated by ?-lactamase (see Table 1 and text).

nalgiovense INBCC103 (commercial starter culture) (?), P. griseofulvum NRRL 2300 liquid culture was performed to confirm that the antibiotic produced by this fungus was penicillin. 2, a peak with a retention time of 13.2 min that corresponds to the control sample of penicillin G was observed in both the unprocessed supernatant and in

the

ethyl

acetate extract. HPLC analysis of the supernatant of a fermentation of P. (A) Control sample of penicillin G at a concentration of 0.1 mg/ml.

The retention time for the penicillin G peak was 13.18 min. (B) Chromatogram of an ethyl acetate-extracted sample of that supernatant. The extraction with ethyl acetate partially purifies and enriches the penicillin G in the sample.

Analysis of the presence of penicillin biosynthetic genes in different strains. The results on production of penicillin observed in the previous experiments should correlate with the presence or absence of the penicillin biosynthetic genes pcbAB , pcbC , and penDE in the different strains.

We performed a series of Southern blot analyses to detect the possible presence of the genes in all the strains used in this study.

The probes used for these experiments are shown in Fig.

Probes

C, D, and E correspond to the pcbAB gene, encoding l -?-aminoadipyl- l -cysteinyl- d -valine (ACV) synthetase, the multidomain enzyme catalyzing the first step of the biosynthesis of penicillin. Probes A and B correspond to the pcbC and penDE genes, respectively, which encode the enzymes responsible for the second and third step of the biosynthesis.



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