Doxycycline and amoxicillin together

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

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

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

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

Probes C, D, lupus and amoxicillin 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. 3) indicate that both the pcbC and the penDE genes are present in all the strains of P.

griseofulvum and are absent in all the remaining strains belonging to other species. The presence of the penicillin gene cluster confirms that P.

Similarly, the absence of the penicillin cluster in P. crustosum supports the conclusion that the antibacterial activity observed in these fungi is not due to penicillin. griseofulvum contained the whole pcbAB gene, as revealed by the hybridization signals appearing with the three probes C, D, and E (Fig.

The Southern blot analyses performed with these five probes, along with additional Southern blots with other probes from the cluster, allowed us to elaborate on an Eco RI restriction map of the new penicillin gene cluster in P. griseofulvum by comparison with the previously described maps of P.

5, there is an Eco RI site in the penDE gene of the P. griseofulvum NRRL 991 strain which is absent in the other strains of P.

In general most of the restriction sites are conserved between the three species, but it is striking that one of the Eco RI fragments inside pcbAB is about 160 bp larger in P. Presence of the genes pcbC and penDE in penicillin-producing strains and absence in nonproducing strains.

Eco RI-digested genomic DNA from all the strains under study was electrophoresed (lanes 1 to 34), transferred to a nylon membrane, and hybridized with probe A (corresponding to the pcbC gene) and probe B (corresponding to the penDE gene). griseofulvum NRRL 2300, NRRL 2152, NRRL 989, NRRL 994, NRRL 991, and NRRL 992; 11 and 12, P.

roqueforti CONT1 (isolated from blue cheese) and NRRL 849; 13, Paecilomyces variotii NRRL 1775; 14 to 17, P. camemberti NRRL 874, NRRL 877, NRRL 876 (ex-type of P. commune NRRL 845 and CEC1 (isolated from cecina); 26, P. Presence of the gene pcbAB in penicillin-producing strains. Eco RI-digested genomic DNA from all the strains was electrophoresed (lanes 1 to 34); transferred to a nylon membrane; and hybridized with probes C, D, and E, which correspond to different portions of the pcbAB gene (Fig. Strain numbers are as indicated in the legend to Fig. verrucosum (lanes 21 and 22) there is a signal of 2.5 kb with probe C, which reveals the presence of the 3? end of the pcbAB gene in this species. With probes D and E, a signal of about 12.5 kb can be observed that corresponds also to the pcbAB gene (see text). Eco RI restriction map of the penicillin gene cluster in P.

The five probes used in the Southern analysis are indicated as grey boxes below the maps of P.

nalgiovense , together with the enzymes used to obtain the probes.

Most Eco RI sites are conserved between the three species, except the site at the 5? end of the penDE gene, which is present only in P. griseofulvum NRRL 991, but in a slightly different position in each of these two strains. An Eco RI fragment inside the pcbAB gene (overlapping partially with probe D) shows a slightly larger size in P.

A similar phenomenon occurs in the right part of the cluster, where the Eco RI fragment comprising the pcbC gene and most of the penDE gene is slightly larger in P. The existence of differences in the restriction map of P. griseofulvum NRRL 991 with respect to the rest of the P.

griseofulvum strains prompted us to confirm by RAPD experiments that all of the P.

griseofulvum collection strains had been correctly identified (Fig.

The pattern of bands obtained with the primers used (see Materials and Methods) clearly indicated that all the strains belonged to the species P. griseofulvum ; therefore, the change in the restriction map of strain NRRL 991 is just a consequence of the genetic variability between P. RAPD analysis of seven strains previously classified as P.

Primers and PCR conditions are indicated in Materials and Methods.

The pattern of bands shown by the different strains clearly indicated that all of them belonged to the species P.

griseofulvum NRRL 2300; 2, NRRL 991; 3, NRRL 992; 4, NRRL 994; 5, NRRL 989; 6, NRRL 2152; 7, P.

verrucosum a striking result was obtained with probes C, D, and E. A 2.5-kb hybridization signal apparently belonging to the pcbAB gene was observed with probe C, which corresponds to the 3? end of pcbAB , whereas with probes D (adjacent to probe C) and E (corresponding to the 5? end of the gene) a signal of about 12.5 kb was found that did not coincide with the Eco RI restriction map of the pcbAB gene of the other species (Fig. The 12.5-kb signal could be explained either by the presence of the pcbAB gene with a different Eco RI restriction map (where only the two Eco RI sites at the 3? end of the gene had been conserved) or by a phenomenon of heterologous hybridization with another gene having a high degree of similarity with the pcbAB gene. To further investigate these possibilities, different DNA fragments from P.

verrucosum genomic DNA were PCR amplified and sequenced. First, we studied whether the 2.5-kb Eco RI fragment located at the 3? end of the pcbAB gene was present in P. verrucosum , as suggested by the result of the hybridization with probe C (Fig. For this purpose the primers NALAB1 and NALAB2, designed according to the sequence of the P.

chrysogenum pcbAB gene (situated at bp 9410 and 10430, respectively, from the ATG) (6), were used to amplify by PCR a DNA fragment of approximately 1 kb from P. verrucosum , included within the 2.5-kb Eco RI fragment. Partial sequence analysis of the amplified fragment showed 95% identity with the P. chrysogenum gene at the DNA level, thus confirming the presence of the 3? end of the pcbAB gene in P. Another pair of primers, PCAB1 and PCAB2, designed also from the P.

chrysogenum pcbAB sequence and belonging to the central part of the gene (located at bp 4497 and 5291, respectively, from the ATG) between probes D and E was used to test the presence of that portion of the pcbAB gene in P.

griseofulvum NRRL 2300 and NRRL 991 were used as control templates in the PCR.

A fragment of the expected size, about 0.8 kb, was amplified with different intensities in all the strains, though in P.

verrucosum additional fragments were also amplified (not shown).

verrucosum 0.8-kb fragment was subcloned and partially sequenced, showing also a very high percentage of identity (96%) at the DNA level to the corresponding fragment of the P.

This result confirmed the presence of the central part of the pcbAB gene (corresponding to the second activating domain of the enzyme) in P. The pcbAB gene encodes ACV synthetase, an enzyme belonging to the family of the nonribosomal peptide synthetases.

These enzymes are organized in domains, each activating one amino acid. In each domain there are several conserved boxes that have different functions in the enzyme (21, 35).

To test whether the hybridization signal of 12.5 kb observed in P.

verrucosum with probes D and E was due to a gene with a high degree of identity with the pcbAB , the following approach was developed.

Two degenerated oligonucleotides, PVCB and PVCG, were designed according to the amino acid sequence of boxes B and G, following the codon usage of P.