Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea

Abstract

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.

FIG. 1.

Species-specific identification of four strains of Arcobacter isolated from samples collected in April and May 2001 by PCR. The gel is an ethidium bromide-stained 1.5% agarose gel containing amplified products generated with primers ArcBUTZ, ArcCRY, and ArcSKIR. Lane 1, 100-bp ladder; lanes 2, 5, 8, and 11, PCR products obtained with A. butzleri-specific primers; lanes 3, 6, 9, and 12, PCR products obtained with A. cryaerophilus-specific primers; lanes 4, 7, 10, and 13, PCR products obtained with A. skirrowii-specific primers; lane 14, PCR product of A butzleri ATCC 49616 DNA amplified with A. butzleri-specific primers; lane 15, PCR product of A. cryaerophilus ATCC 43157 DNA amplified with A. cryaerophilus-specific primers; lane 16, PCR product of A. skirrowii ATCC 51132 DNA amplified with A. skirrowii-specific primers.

FIG. 2.

PCR products amplified from a single seawater sample obtained in March 2002 after different extraction methods were used. The gel is an ethidium bromide-stained 1.5% agarose gel containing amplified products generated with primers ArcBUTZ, ArcCRY, and ArcSKIR. Lane 1, 100-bp ladder; lanes 2 to 4, extraction method A; lanes 5 to 7, extraction method B; lanes 8 to 10, extraction method C; lanes 11 to 13, extraction method D; lanes 2, 5, 8, and 11, PCR products obtained with A. butzleri-specific primers; lanes 3, 6, 9, and 12, PCR products obtained with A. cryaerophilus-specific primers; lanes 4 and 7, PCR products obtained with A. skirrowii-specific primers; lanes 10 and 13, PCR products obtained with A. skirrowii-specific primers; lane 14, PCR product of A butzleri ATCC 49616 DNA amplified with A. butzleri-specific primers; lane 15, PCR product of A. cryaerophilus ATCC 43157 DNA amplified with A. cryaerophilus-specific primers; lane 16, PCR product of A. skirrowii ATCC 51132 DNA amplified with A. skirrowii-specific primers.

FIG. 3.

PCR products amplified from a single small-plankton sample obtained in December 2001 after different extraction methods were used. The gel is an ethidium bromide-stained 1.5% agarose gel containing amplified products generated with primers ArcBUTZ, ArcCRY, and ArcSKIR. Lane 1, 100-bp ladder; lanes 2 to 4, extraction method A; lanes 5 to 7, extraction method B; lanes 8 to 10, extraction method C; lanes 11 to 13, extraction method D; lanes 2, 5, and 8, PCR products obtained with A. butzleri-specific primers; lanes 3, 6, and 9, PCR products obtained with A. cryaerophilus-specific primers; lane 11, PCR products obtained with A. butzleri-specific primers; lane 12, PCR products obtained with A. cryaerophilus-specific primers; lanes 4, 7, 10, and 13, PCR products obtained with A. skirrowii-specific primers; lane 14, PCR product of A butzleri ATCC 49616 DNA amplified with A. butzleri-specific primers; lane 15, PCR product of A. cryaerophilus ATCC 43157 DNA amplified with A. cryaerophilus-specific primers; lane 16, PCR product of A. skirrowii ATCC 51132 DNA amplified with A. skirrowii-specific primers.

FIG. 4.

PCR products amplified from a single large-plankton sample obtained in December 2001 after different extraction methods were used. The gel is an ethidium bromide-stained 1.5% agarose gel containing amplified products generated with primers ArcBUTZ, ArcCRY, and ArcSKIR. Lane 1, 100-bp ladder; lanes 2 to 4, extraction method A; lanes 5 to 7, extraction method B; lanes 8 to 10, extraction method C; lanes 11 to 13, extraction method D; lanes 2, 5, 8, and 11, PCR products obtained with A. butzleri-specific primers; lanes 3, 6, 9, and 12, PCR products obtained with A. cryaerophilus-specific primers; lanes 4, 7, 10, and 13, PCR products obtained with A. skirrowii-specific primers; lane 14, PCR product of A butzleri ATCC 49616 DNA amplified with A. butzleri-specific primers; lane 15, PCR product of A. cryaerophilus ATCC 43157 DNA amplified with A. cryaerophilus-specific primers; lane 16, PCR product of A. skirrowii ATCC 51132 DNA amplified with A. skirrowii-specific primers.