Detection of Helicobacter pylori associated with zooplankton

Abstract

Helicobacter pylori was isolated from marine zooplankton, and characterized by standard microbiological tests, by PCR amplification of vacA and cagA gene fragments, and by comparative sequence analysis of the vacA PCR product. In a viable-but-non-culturable (VBNC) state, this isolate, as well as the reference strain H. pylori ATCC 43629, could be re-activated only when incubated in the presence of the marine copepod Tigriopus fulvus, and not in its absence. Isolate and type strain of H. pylori were found to be associated to the surface of T. fulvus, which supports speculations about a potential role of copepods in H. pylori survival and transmission.KEY WORDS: Helicobacter pylori · Zooplanktonic organisms · Transmission · Seawater · Viable-butnon-culturable (VBNC) state · Tigriopus fulvus Resale or republication not permitted without written consent of the publisherAquat Microb Ecol 40: [115][116][117][118][119][120] 2005 pylori cells from seawater samples and associated to zooplankton. An isolate and the type strain of H. pylori were subsequently used to study potential interactions with zooplankton. For this purpose, the marine copepod Tigriopus fulvus (Fisher 1860) found in rockpools of the Ligurian coast (Carli et al. 1993) was used as a model organism. MATERIALS AND METHODSWater sampling and processing. Water samples of 10 l were collected with a Niskin bottle at a depth of 5 m from the Adriatic Sea (42°29' 41'' N, 14°12' 19'' E), about 500 m from the coast of Pescara, Italy. Water samples were refrigerated at 4°C and processed within 4 h. They were first passed through a 200 µm mesh nylon filter (Idromar). Filters with retained organisms were transferred to sterile plastic containers, and covered with 10 ml of sterile seawater and sterile glassbeads. After detachment of organisms from the filter by a minishaker (IKA WORKS) at 600 rpm for 1 min, the samples were twice centrifuged with sterile seawater at 200 × g for 10 min to remove free bacteria, and the remaining organisms finally resuspended in 10 ml of sterile seawater.The filtered water was subsequently passed through a 64 µm mesh nylon filter, which was subsequently treated as described above. The remaining filtered water was finally passed through 0.22 µm pore-size standard membrane filters (Pall Gelman Laboratory) to detect free cells of Helicobacter pylori and the filter was treated as above. From each sample, 3 equal aliquots were prepared before culture processing.Isolation and characterization of Helicobacter pylori. From samples obtained on these different filters, 100 µl were directly spread on non-selective medium with 10% of defibrinate horse blood (Chocolate agar, CA, OXOID) supplemented with 1% of IsoVitaleX (Becton Dickinson), and on Campylobacter selective medium (CP, OXOID) and incubated at 37°C under microaerophilic conditions (Campy Pak Jar, OXOID) for 5 to 7 d. Plates were checked every day and potential colonies of Helicobacter pylori were subcultured on CA and CP under microaerophilic conditions. The remaining sea wat...

Failure To Detect Helicobacter pylori DNA in Drinking and Environmental Water in Dhaka, Bangladesh, Using Highly Sensitive Real-Time PCR Assays

“…In addition, there are a number of studies reporting traces of H. pylori in different water sources, mainly using PCR-based methods, although successful isolation of live H. pylori from river water (33) or marine zooplankton (10) have been reported. However, some of these studies were performed with river water, lake water, or seawater (16, 33, 43, 49) rather than drinking water, and some used nested PCR (34,40,43), which may increase detection sensitivity but is associated with an increased risk for contamination.…”

Abundance and Distribution of Enteric Bacteria and Viruses in Coastal and Estuarine Sediments—a Review

“…exhibited a reduction in nutrient transport, respiration rates and macromolecular synthesis compared to culturable equivalents; however these VBNC cells can still actively divide at a reduced rate (Peneau et al, 2007). Adhesion to the external surface of zooplankton also stimulates fecal enterococci to enter a VBNC state (Signoretto et al, 2004) and this may form a vital part of the transmission pathway (Cellini et al, 2005). Favorable growth conditions and an ideal stoichiometric ratio of carbon to inorganic elements enables recovery from VBNC state, although the resuscitation rate is highly variable depending on species and conditions studied (Arana et al, 2007; Bari et al, 2013; Ayrapetyan et al, 2014a) and may take days to occur (Scherber et al, 2009).…”

Bacteria associated with copepods: abundance, activity and community composition

“…Most previous studies have focused on copepod associated bacterial communities as reservoirs of specific bacterial pathogens (e.g. Cellini et al 2005, Huq et al 2005 and on specific bacterial groups. For instance, Heidelberg et al (2002) found that Vibrio species constituted a large proportion of the bacteria associated with copepods in Chesapeake Bay.…”