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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.

Dosage is given twice bacterial findings in acute eyelids, which may occur in only one eye Other causes include antibiotics (particularly neomycin) and the preservative thiomersal (a preservative in eye.

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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 amoxicillin 500 tablet 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. With these primers, a temperature gradient PCR was performed. Several bands amplified; among these, one of the expected size for the pcbAB gene domains (1.0 kb) became predominant at higher temperatures. This band when subcloned was shown to be composed of two different DNA fragments, which corresponded, respectively, to positions 1190 to 2197 and 4472 to 5467, numbered from the P. The sequence of these fragments revealed that they corresponded, respectively, to the first and

second

activating domains of the ACV synthetase, and showed a high degree (92 to 96%) of identity at the DNA level with the pcbAB gene of P. Eleven other amplified fragments of different sizes were subcloned and sequenced. Their sequences did not show significant identity with peptide synthetases.

Therefore, we concluded that the 12.5-kb hybridization signal in P. verrucosum corresponds to the pcbAB gene and that this gene is present apparently in all its length in this fungus; no evidence for the possible presence of additional peptide synthetase genes in P. Among different fungal species belonging to the genus Penicillium that are currently used as starter cultures in the food industry or that are frequently isolated from cured meat products, only P.

griseofulvum (in addition to the previously reported P. nalgiovense ) was found to produce penicillin and possess the three penicillin biosynthetic genes ( pcbAB , pcbC , and penDE ). It is important that the commonly used cheese starters P. roqueforti neither are penicillin producers nor possess the penicillin biosynthetic genes, which implies that they do not represent a risk regarding the problem of the presence of penicillin in food.

griseofulvum is frequently isolated from corn, wheat, barley, flour, and walnuts (40) and from meat products (27), thus being a potential source for the presence of penicillin in food. griseofulvum strains used in the present study was sensitive to ?-lactamase, indicating that P. griseofulvum is a penicillin producer, which was confirmed by HPLC (Fig.

griseofulvum strains—NRRL 991, NRRL 992, and NRRL 989—an antibacterial activity could still be detected in the presence of excess ?-lactamase, suggesting that another compound with antibacterial activity must be produced and secreted to the medium in these strains.



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