Salmonella spp., Vibrio spp., Clostridium perfringens, and Plesiomonas shigelloides in Marine and Freshwater Invertebrates from Coastal California Ecosystems

The coastal ecosystems of California are highly utilized by humans and animals, but the ecology of fecal bacteria at the land–sea interface is not well understood. This study evaluated the distribution of potentially pathogenic bacteria in invertebrates from linked marine, estuarine, and freshwater ecosystems in central California. A variety of filter-feeding clams, mussels, worms, and crab tissues were selectively cultured for Salmonella spp., Campylobacter spp., Escherichia coli-O157, Clostridium perfringens, Plesiomonas shigelloides, and Vibrio spp. A longitudinal study assessed environmental risk factors for detecting these bacterial species in sentinel mussel batches. Putative risk factors included mussel collection near higher risk areas for livestock or human sewage exposure, adjacent human population density, season, recent precipitation, water temperature, water type, bivalve type, and freshwater outflow exposure. Bacteria detected in invertebrates included Salmonella spp., C. perfringens, P. shigelloides, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio alginolyticus. Overall, 80% of mussel batches were culture positive for at least one of the bacterial species, although the pathogens Campylobacter, E. coli-O157, and Salmonella were not detected. Many of the same bacterial species were also cultured from upstream estuarine and riverine invertebrates. Exposure to human sewage sources, recent precipitation, and water temperature were significant risk factors for bacterial detection in sentinel mussel batches. These findings are consistent with the hypothesis that filter-feeding invertebrates along the coast concentrate fecal bacteria flowing from land to sea and show that the relationships between anthropogenic effects on coastal ecosystems and the environmental niches of fecal bacteria are complex and dynamic.     

Campylobacter spp., Enterococcus spp., Escherichia coli, Salmonella spp., Yersinia spp., and Cryptosporidium oocysts in semi-domesticated reindeer (Rangifer tarandus tarandus) in Northern Finland and Norway

The specific aim of this study was to assess the faecal shedding of zoonotic enteropathogens by semi-domesticated reindeer (Rangifer tarandus tarandus) to deduce the potential risk to human health through modern reindeer herding. In total, 2,243 faecal samples of reindeer from northern regions of Finland and Norway were examined for potentially enteropathogenic bacteria (Campylobacter species, Enterococcus species, Escherichia coli, Salmonella species and Yersinia species) and parasites (Cryptosporidium species) in accordance with standard procedures. Escherichia coli were isolated in 94.7%, Enterococcus species in 92.9%, Yersinia species in 4.8% of the samples and Campylobacter species in one sample only (0.04%). Analysis for virulence factors in E. coli and Yersinia species revealed no pathogenic strains. Neither Salmonella species nor Cryptosporidium oocysts were detected. The public health risk due to reindeer husbandry concerning zoonotic diseases included in this study has to be considered as very low at present but a putative epidemiological threat may arise when herding conditions are changed with respect to intensification and crowding.     

Staphylococcus saprophyticus is a pathogen of the cattle tick Rhipicephalus (Boophilus) microplus

During experimental Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) infestation on cattle, approximately 5% of engorged female ticks showed symptoms of bacterial infection. The affected ticks were unable to oviposit, secreted a distinctive yellow exudate through the genital orifice and eventually died. Microscopic analysis of tick exudate showed abundant clusters of Gram-positive cocci bacteria that were isolated and cultured on bacteriological medium. Biochemical phenotyping and 16S rRNA ribotyping analysis on cultured bacteria identified it as Staphylococcus saprophyticus. This species was also isolated from healthy tick larvae, indicating that S. saprophyticus is commonly found in ticks during different developmental stages. However, conspicuous symptoms are only found on fully engorged females. Cultured S. saprophyticus induced identical pathological symptoms when the bacteria were experimentally inoculated into healthy ticks, demonstrating it to be the causative agent of the R. microplus infectious lethal disease described in this work.     

Detection of Rickettsia and Anaplasma from hard ticks in Thailand

We collected a total of 169 adult hard ticks and 120 nymphs from under the leaves of plants located along tourist nature trails in ten localities. The results present data examining the vector competence of ticks of different genera and the presence of Rickettsia and Anaplasma species. The ticks belonged to three genera, Amblyomma, Dermacentor, and Haemaphysalis, comprising 11 species. Rickettsia bacteria were detected at three collection sites, while Anaplasma bacteria were detected at only one site. Phylogenetic analysis revealed new rickettsia genotypes from Thailand that were closely related to Rickettsia tamurae, Rickettsia monacensis, and Rickettsia montana. This study was also the first to show that Anaplasma bacteria are found in Haemaphysalis shimoga ticks and are closely related evolutionarily to Anaplasma bovis. These results provide additional information for the geographical distribution of tick species and tick‐borne bacteria in Thailand and can therefore be applied for ecotourism management.     

First report of lyme disease spirochetes in ticks from Romania (Sibiu County)

We examined 200 questing Ixodes ricinus ticks (nymphs and adults) collected in three different sites in Sibiu County, Romania by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for Borrelia burgdorferi s.l.. We detected the bacteria in 19% of the investigated ticks. The prevalence of infection was higher in nymphs (27%) than in adults (12%). Two B. burgdorferi sensu lato genospecies were detected: B. garinii (20%) and B. afzelii (80%). We did not detect any mixed infections in the investigated ticks. In two of the investigated sites B. burgdorferi prevalence values were comparable (25%), while in the third site the prevalence was lower (≈7%). Our preliminary study provides evidence that Lyme disease spirochetes are present in various areas and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects that undergo work or leisure activities in those areas.     

Prevalence of Anaplasma and Bartonella spp. in Ticks Collected from Korean Water Deer (Hydropotes inermis argyropus)

Deer serve as reservoirs of tick-borne pathogens that impact on medical and veterinary health worldwide. In the Republic of Korea, the population of Korean water deer (KWD, Hydropotes inermis argyropus) has greatly increased from 1982 to 2011, in part, as a result of reforestation programs established following the Korean War when much of the land was barren of trees. Eighty seven Haemaphysalis flava, 228 Haemaphysalis longicornis, 8 Ixodes nipponensis, and 40 Ixodes persulcatus (21 larvae, 114 nymphs, and 228 adults) were collected from 27 out of 70 KWD. A total of 89/363 ticks (266 pools, 24.5% minimum infection rate) and 5 (1.4%) fed ticks were positive for Anaplasma phagocytophilum using nested PCR targeting the 16S rRNA and groEL genes, respectively. The 16S rRNA gene fragment sequences of 88/89 (98.9%) of positive samples for A. phagocytophilum corresponded to previously described gene sequences from KWD spleen tissues. The 16S rRNA gene fragment sequences of 20/363 (5.5%) of the ticks were positive for A. bovis and were identical to previously reported sequences. Using the ITS specific nested PCR, 11/363 (3.0%) of the ticks were positive for Bartonella spp. This is the first report of Anaplasma and Bartonella spp. detected in ticks collected from KWD, suggesting that ticks are vectors of Anaplasma and Bartonella spp. between reservoir hosts in natural surroundings.     

Increased Abundance of Gallionella spp., Leptothrix spp. and Total Bacteria in Response to Enhanced Mn and Fe Concentrations in a Disturbed Southern Appalachian High Elevation Wetland

The Sorrento wetland hosts several Fe- and Mn-rich seeps that are reported to have appeared after the area was disturbed by recent attempts at development. Culture-independent and culture-based analyses were utilized to characterize the microbial community at the main site of the Fe and Mn seep. Several bacteria capable of oxidizing Mn(II) were isolated, including members related to the genera Bacillus, Lysinibacillus, Pseudomonas, and Leptothrix, but none of these were detected in clone libraries. Most probable number assays demonstrated that seep and wetland sites contained higher numbers of culturable Mn-oxidizing microorganisms than an upstream reference site. When compared with quantitative real time PCR (qPCR) assays of total bacteria, MPN analyses indicated that less than 0.01% of the total population (estimated around 10⁹ cells/g) was culturable. Light microscopy and fluorescence in situ hybridization (FISH) images revealed an abundance of morphotypes similar to Fe- and Mn-oxidizing Leptothrix spp. and Gallionella spp. in seep and wetland sites. FISH allowed identification of Leptothrix-type sheath-forming organisms in seep samples but not in reference samples. Gallionella spp. and Leptothrix spp. cells numbers were estimated using qPCR with a novel primer set that we designed. Results indicated that numbers of Gallionella, Leptothrix or total bacteria were all significantly higher at the seep site relative to the reference site (where Gallionella was below detection). Interestingly, numbers of Leptothrix in the seep site were estimated at only 10⁷ cells/g and were not statistically different in the late summer versus the late winter, despite dramatic changes in sheath abundance (as indicated by microscopy). qPCR also indicated that Gallionella spp. may represent up to 10% (3 × 10⁸ cells/g) of the total bacteria in seep samples. These data corroborate clone library data from samples taken in October 2008, where 11 SSU rRNA sequences related to Gallionella spp. were detected out of 77 total sequences (roughly 10–15%), and where Leptothrix sequences were not detected. Analysis of this SSU rRNA clonal library revealed that a diverse microbial community was present at seep sites. At a 3% difference cutoff, 30 different operational taxonomic units were detected out of 77 sequences analyzed. Dominant sequence types clustered among the beta- and gamma- Proteobacteria near sequences related to the genera Ideonella, Rhodoferax, Methylotenera, Methylobacter, and Gallionella. Overall, results suggest that high metal concentrations at the seep sites have enriched for Fe- and Mn-oxidizing bacteria including organisms related to Gallionella and Leptothrix species, and that members of these genera coexist within a diverse microbial community.     

Phage specificity and lipopolysaccarides of stem- and root-nodulating bacteria (Azorhizobium caulinodans, Sinorhizobium spp., and Rhizobium spp.) of Sesbania spp

Phage susceptibility pattern and its correlation with lipopolysaccharide (LPS) and plasmid profiles may help in understanding the phenotypic and genotypic diversity among highly promiscuous group of rhizobia nodulating Sesbania spp.; 43 phages were from two stem-nodulating bacteria of S. rostrata and 16 phages were from root-nodulating bacteria of S. sesban, S. aegyptica and S. rostrata. Phage susceptibility pattern of 38 Sesbania nodulating bacteria was correlated with their LPS rather than plasmid profiles. Different species of bacteria (A. caulinodans- ORS571, SRS1-3 and Sinorhizobium saheli- SRR907, SRR912) showing distinct LPS subtypes were susceptible to different group of phages. Phages could also discriminate the strains of Si. saheli (SSR312, SAR610) possessing distinct LPS subtypes. Phages of Si. meliloti (SSR302) were strain-specific. All the strains of R. huautlense having incomplete LPS (insignificant O-chain) were phage-resistant. In in vitro assay, 100% of the phages were adsorbed to LPS of indicator bacterium or its closely related strain(s) only. These observations suggest the significance of LPS in phage specificity of Sesbania nodulating rhizobia. Highly specific phages may serve as biological marker for monitoring the susceptible bacterial strains in culture collections and environment.     

Molecular detection of Rickettsia aeschlimannii in Hyalomma spp. ticks from camels (Camelus dromedarius) in Nigeria,West Africa

Several species of the spotted fever group rickettsiae have been identified as emerging pathogens throughout the world, including in Africa. In this study, 197 Hyalomma ticks (Ixodida: Ixodidae) collected from 51 camels (Camelus dromedarius) in Kano, northern Nigeria, were screened by amplification and sequencing of the citrate synthase (gltA), outer membrane protein A (ompA) and 17‐kDa antigen gene fragments. Rickettsia sp. gltA fragments were detected in 43.3% (42/97) of the tick pools tested. Rickettsial ompA gene fragments (189 bp and 630 bp) were detected in 64.3% (n = 27) and 23.8% (n = 10) of the gltA‐positive tick pools by real‐time and conventional polymerase chain reaction (PCR), respectively. The amplicons were 99–100% identical to Rickettsia aeschlimannii TR/Orkun‐H and R. aeschlimannii strain EgyRickHimp‐El‐Arish in GenBank. Furthermore, 17‐kDa antigen gene fragments of 214 bp and 265 bp were detected in 59.5% (n = 25) and 38.1% (n = 16), respectively, of tick pools, and sequences were identical to one another and 99–100% identical to those of the R. aeschlimannii strain Ibadan A1 in GenBank. None of the Hyalomma impressum ticks collected were positive for Rickettsia sp. DNA. Rickettsia sp. gltA fragments (133 bp) were detected in 18.8% of camel blood samples, but all samples were negative for the other genes targeted. This is the first report to describe the molecular detection of R. aeschlimannii in Hyalomma spp. ticks from camels in Nigeria.     

Rickettsia spp., Ehrlichia sp. and Candidatus Midichloria sp. associated to ticks from a protected urban area in Buenos Aires City (Argentina)

The aim of this study was to determine the infection with Rickettsiales in ticks and birds from the main protected urban area of Buenos Aires City (Argentina). One Amblyomma aureolatum (0.2%) and one Ixodes auritulus (0.1%) were positive by PCR targeting Rickettsia 23S-5S rRNA intergenic spacer. Phylogenetic analysis shows to findings in A. aureolatum are closely to Rickettsia bellii and for I. auritulus are related to ‘Candidatus Rickettsia mendelii’. One I. auritulus (0.1%) and three A. aureolatum (0.6%) were positive by PCR for a fragment of the 16S rRNA gene of the Anaplasmataceae family. The sequences obtained from A. aureolatum were phylogenetically related to Midichloriaceae endosymbionts. The sequence from I. auritulus s.l. had 100% identity with Ehrlichia sp. Magellanica from Chile and two genotypes of Ehrlichia sp. from Uruguay. The results of our study show that Rickettsia and Ehrlichia are present in ticks in the main protected urban area of Buenos Aires City.

     

Studies on the critical water mass and the rehydration potential of unfed adult Dermacentor marginatus and D. reticulatus ticks (Acari: Ixodidae)

In unfed adult Dermacentor marginatus and D. reticulatusticks survival and capability to restore water balance after loss of high percentages of exchangeable body water were investigated. Furthermore, it was examined how frequently dehydrated ticks of these species were able to rehydrate by uptake of atmospheric water vapour. The critical water mass, defined as the water mass remaining in a tick at the nonambulatory state, differed between light and heavy weight groups and averaged 62.4 and 55.8%, respectively, of the total body water of fully hydrated ticks in females, and 54.4 and 51.1% respectively, in males of D. marginatus. In D. reticulatus, the corresponding figures were 55.9 and 54.7% in females and 52.1 and 52.7% in males. All ticks survived dehydration to 50, 75 or 100% of the critical water mass, and 96.7% of the D. marginatus ticks and 95.8% of the D. reticulatus ticks compensated water losses during subsequent incubation at 95% relative humidity (r.h.) and 20°C. Unfed females and males of both Dermacentor spp. were capable to balance water loss very frequently over a period of several months. When ticks were repeatedly dehydrated at 0% r.h. for 7 days and rehydrated at 95% r.h. and 20°C, females and males of D. marginatus reached the 50% mortality after 22 and 29 cycles of de- and rehydration, respectively, during 211 and 285 days, respectively. In D. reticulatus, 50% of females and males survived 23 and 17 cycles, respectively, during 248 and 186 days, respectively. Rehydration weights were as high or even higher as those of ticks kept at permanent 95% r.h.     

Studies on survival and water balance of unfed adult Dermacentor marginatus and D. reticulatus ticks (Acari: Ixodidae)

The water content, the survival time at various relative humidities(r.h.) and the critical equilibrium activity of unfed adultDermacentor marginatus and D.reticulatus ticks were investigated at a constant temperature of20 °C. It was also examined whether these ticks use liquidwaterto compensate water loss. Both Dermacentor spp. showed nosignificant differences in water content in relation to body mass. The meanwater content of D. marginatus and D.reticulatus was 54.6% and 54.7%, respectively, in females and 56.3%and 57.0%, respectively, in males. The survival time of unfed adults prolongedwith decreasing saturation deficits. On average, males survived longer thanfemales and D. marginatus ticks survived mostly longerthanD. reticulatus ticks. The 50% mortality period rangedbetween 40 d at 33% r.h. and 420 d at 95% r.h. in D.marginatus, and between 43 d at 33 r.h. and 366 d at 95% r.h. inD. reticulatus. The critical equilibrium activity of unfedadults was estimated to be 0.84 for both species and was independent of sex.When dehydrated adult D. marginatus and D.reticulatus ticks were offered liquid water, only a few slightlygained weight while most further lost weight. Liquid water was not attractivefor dehydrated or non-dehydrated ticks and drinking was not observed. Aftersubmerging in water for 2 d, most of the dehydrated ticks had gained weight.     

Infestation of taiga ticks with borrelias in the territory of Novosibirsk Scientific Center (Siberian Branch,Russian Academy of Sciences)

Borrelia specimens were revealed in taiga ticks Ixodes persulcatus collected in the wild by flagging and also in ticks provided by the Vaccination section of the Novosibirsk Scientific Center, Siberian Branch of the Russian Academy of Sciences (NSC); these ticks were obtained from patients attacked by ticks. Isolation of borrelias in the BSK-H medium had demonstrated the presence of B. garinii, B. afzelii, and B. miyamotoi in the territory of NCS. B. miyamotoi isolates were unstable, loosing their growth ability during subsequent cultivation. DNA of the three above species was detected by PCR in tick samples collected by flagging and obtained from humans. DNA of B. garinii was recorded in ticks more often; DNA of B. afzelii was found less frequently; B. miyamotoi DNA was detected in the smallest number of ticks. In ticks collected by flagging, DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 38.6%, 9.9%, and in 3.9% of specimens, respectively. In ticks collected from attacked humans, the number of positive tests was lower; e.g., DNA of B. garinii, B. afzelii, and B. miyamotoi was detected in 24.2%, 6.9%, and in 5.6% of samples, respectively. Mixed infection of ticks with two Borrelia species was also detected; DNA of B. miyamotoi and of B. garinii was detected in mixed infections more frequently.     

Ixodid ticks of road-killed wildlife species in southern Italy: new tick-host associations and locality records

In this study 1,868 questing Ixodes ricinus ticks (nymphs and adults), collected in six sites from three counties—Giurgiu, Sibiu, and Tulcea—in Romania, were examined by polymerase chain reaction (PCR) followed by reverse line blot (RLB) for detection of Borrelia burgdorferi sensu lato presence. The bacteria were found in 18% of the investigated ticks. The prevalence of infection did not differ significantly between nymphs (19.1%) and adults (15.4%). Three B. burgdorferi s.l. genospecies were detected: B. afzelii (61.1%), B. garinii (31.2%), and B. valaisiana (7.7%). No mixed infections were detected. The highest infection prevalence in nymphs was detected at Cristian (Sibiu County)—22.0%, whereas in adults it was at Comana (Giurgiu County)—19.8%. This preliminary study provides evidence that Lyme disease spirochetes are present in various regions of Romania, and at a relatively high prevalence in their vectors, thus posing a risk of infection to human subjects in the areas infested by ticks.     

Detection of spotted fever group Rickettsia spp. from bird ticks in the U.K.

Migratory birds are known to play a role in the long‐distance transportation of microorganisms. To investigate whether this is true for rickettsial agents, we undertook a study to characterize tick infestation in populations of the migratory passerine bird Riparia riparia (Passeriformes: Hirundinidae), the sand martin. A total of 194 birds were sampled and ticks removed from infested birds. The ticks were identified as female Ixodes lividus (Acari: Ixodidae) using standard morphological and molecular techniques. Tick DNA was assayed to detect Rickettsia spp. using polymerase chain reaction and DNA was sequenced for species identification. A single Rickettsia spp. was detected in 100% of the ticks and was designated Rickettsia sp. IXLI1. Partial sequences of 17‐kDa and ompA genes showed greatest similarity to Rickettsia sp. TCM1, an aetiological agent of Japanese spotted fever‐like illness, previously described in Thailand. Phylogenetic analysis showed that Rickettsia sp. IXLI1 fitted neatly into a group containing strains Rickettsia japonica, Rickettsia sp. strain Davousti and Rickettsia heilongjiangensis. In conclusion, this research shows that U.K. migratory passerine birds host ticks infected with Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents.     

Detection of Bartonella DNA in roe deer (Capreolus capreolus) and in ticks removed from deer

The potential role of roe deer as a sylvatic reservoir of Bartonella in north-west Poland has been assessed. In addition, ticks infesting roe deer were screened to assess their role as a vector and reservoir of Bartonella. Blood and tissue samples of 72 animals from north-western Poland were PCR-screened. Bartonella DNA was detected by using primers complementary to the intergenic spacer between the 16S and 23S rRNA genes, which is used for identification of over a dozen species of this genus. Products of three different sizes were detected: 230 and 290 bp, representative of two strains of Bartonella capreoli, and 190 bp, identified as Bartonella bovis. All the three amplicons were detected in the blood, spleen and liver from the roe deer. All samples from the heart, lungs and kidneys were PCR negative. In ticks (Ixodes ricinus), only the 290 bp fragment from B. capreoli was present. Generally, Bartonella infection rate in Capreolus capreolus amounted to 27.6% of the roe deer, but it was much higher during winter (62%) than in spring (6.9%). The results show that the roe deer may be a reservoir for B. capreoli and B. bovis. The infection detected in I. ricinus ticks (7.7%) suggests that ticks may act as a Bartonella reservoir and vector.     

The occurrence of Pseudomonas spp. in surface water and in tap water as determined on citrate media

Citrate-utilizing bacteria were counted in 289 samples of tap water derived from either surface water or ground water and in 32 samples of raw or partially treated surface water by using media containing ferric ammonium citrate as the carbon and energy source.The citrate-utilizing bacteria constituted only small minorities of the colony counts on Lab-Lemco agar at 25 C in both tap water and surface water.A total of 1071 isolates were obtained, of which 979 were able to utilize citrate. Characterization of the citrate-utilizing isolates revealed that 90% of these bacteria were arginine dihydrolase-positive and belonged to the genera Pseudomonas (84.3%) and Aeromonas (5.7%).The genus Pseudomonas was represented by fluorescent pseudomonads (66.3%), non-fluorescent glucose-utilizing pseudomonads (12.1%) and P. alcaligenes (5.9%). None of the isolates was identified as P. aeruginosa.It is suggested that the pseudomonads and the aeromonads are not adapted to low substrate concentrations. Enumeration of the citrate-utilizing bacteria in tap water therefore may give information on the efficiency of water treatment techniques as regards the substrate removal.     

Borrelia burgdorferi sensu lato and co‐infections with Anaplasma phagocytophilum and Rickettsia spp. in Ixodes ricinus in Hamburg,Germany

To obtain initial data on Borrelia burgdorferi sensu lato (Spirochaetales: Spirochaetaceae) in Ixodes ricinus (Ixodida: Ixodidae) ticks in Hamburg, Germany, 1400 questing ticks were collected by flagging at 10 different public recreation areas in 2011 and analysed using probe‐based quantitative real‐time polymerase chain reaction. The overall rate of infection with B. burgdorferi s.l. was 34.1%; 30.0% of adults were infected (36.7% of females and 26.0% of males), as were 34.5% of nymphs. Significant differences in tick infection rates were observed between the spring and summer/autumn months, as well as among sampling locations. Borrelia genospecies identification by reverse line blotting was successful in 43.6% of positive tick samples. The most frequent genospecies was Borrelia garinii/Borrelia bavariensis, followed by Borrelia afzelii, Borrelia valaisiana, B. burgdorferi sensu stricto, Borrelia spielmanii, Borrelia bissettii and Borrelia lusitaniae. Based on previously published data, co‐infection of Borrelia and Rickettsiales spp. was determined in 25.8% of ticks. Overall, 22.9% of ticks were co‐infected with Rickettsia spp. (Rickettsiales: Rickettsiaceae), 1.7% with Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), and 1.2% with both pathogens. Study results show a high prevalence of Borrelia‐positive ticks in recreation areas in the northern German city of Hamburg and the potential health risk to humans in these areas should not be underestimated.     

Hyphomonas spp., Shewanella spp., and Other Marine Bacteria Lack Heterogeneous (Ladderlike) Lipopolysaccharides

The lipopolysaccharides (LPS) of 19 marine bacteria were examined for size heterogeneities by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in conjunction with an LPS-specific silver staining method. Fifteen marine bacteria had an R-type LPS instead of the ladderlike LPS array characteristic of most bacteria. In addition, three marine bacteria also had a single large LPS molecule. Without constraints (e.g., surface masking), R-type LPS, a more hydrophobic molecule, predominates in Shewanella and Hyphomonas species and in other marine bacteria.     

Molecular monitoring of Escherichia coli O157: H7 sterilization rate using qPCR and propidium monoazide treatment

Propidium monoazide is a DNA‐intercalating dye. PMA‐qPCR has been reported as a novel method to detect live bacteria in complex samples. In this study, this method was used to monitor the sterilization effects of UHP, ultrasound and high PEF on Escherichia coli O157:H7. Our results showed that all three sterilization techniques are successful to kill viable E. coli O157:H7 cells under their appropriate conditions. PMA‐qPCR can effectively monitor the amount of DNA released from viable E. coli O157:H7 cells, and the results from PMA‐qPCR were highly consistent with those from plate counting after treatment with UHP, ultrasound and high PEF. The maximal ΔC_t between PMA‐qPCR and qPCR obtained in this study was 10·39 for UHP, 5·76 for ultrasound and 2·30 for high PEF. The maximal sterilization rates monitored by PMA‐qPCR were 99·92% for UHP, 99·99% for ultrasound and 100% for high PEF. Thus, PMA‐qPCR can be used to detect the sterilization effect on food and water supplies after treatment with UHP, ultrasound and high PEF.

Significance and Impact of the Study

The reliable detection of viable foodborne pathogenic bacteria in water and food is of great importance in our daily life. However, the traditional bacteria cultivation‐based methods are time‐consuming and difficult to monitor all viable bacteria because of the limitation of cultivation conditions. This study demonstrated that PMA‐qPCR technique is very effective to monitor viable E. coli O157:H7 after sterilization and will help to monitor the viable bacteria in food and water.