Enhancement of viable Campylobacter detection by chemotactic stimuli

The effects of chemotactic stimuli on motility ability of viable Campylobacter to pass through a 0.45 µm pore size filter in viscous condition were investigated. Reference strains including C. jejuni ATCC 33291, C. coli MUMT 18407, C. lari ATCC 43675, and C. upsaliensis DMST 19055 were used. The initial numbers of artificially-inoculated viable cells per g of chicken meat were approximately 10 to 10⁴. Constituents of mucin plus bile (1:1), varieties of amino acids, and sodium salts were added into a soft-agar-coated membrane filter and incubated at both 37 °C and 42 °C for 24 h. The drop plate method was used to determine numbers of viable Campylobacter at 6, 12, 18, and 24 h. After 6 h, constituents of mucin plus bile at the concentrations of 1, 5, and 10% demonstrated significant increases in numbers of viable cells (p < 0.05). The numbers of the organisms at 42 °C were higher than those at 37 °C. In contrast, no significant difference in cell numbers was observed by adding amino acids or sodium salts. In addition, the role of starvation on chemotactic responses was also studied. Starved cells showed lower chemotactic response than non-starved cells. This method permitted rapid detection of viable thermophilic Campylobacter.     

Antimicrobial resistance in Campylobacter: Susceptibility testing methods and resistance trends

Most Campylobacter infections are self-limiting but antimicrobial treatment (e.g., macrolides, fluoroquinolones) is necessary in severe or prolonged cases. Susceptibility testing continues to play a critical role in guiding therapy and epidemiological monitoring of resistance. The methods of choice for Campylobacter recommended by the Clinical and Laboratory Standards Institute (CLSI) are agar dilution and broth microdilution, while a disk diffusion method was recently standardized by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Macrolides, quinolones, and tetracyclines are among the common antimicrobials recommended for testing. Molecular determination of Campylobacter resistance via DNA sequencing or PCR-based methods has been performed. High levels of resistance to tetracycline and ciprofloxacin are frequently reported by many national surveillance programs, but resistance to erythromycin and gentamicin in Campylobacter jejuni remains low. Nonetheless, variations in susceptibility observed over time underscore the need for continued public health monitoring of Campylobacter resistance from humans, animals, and food.     

Amoebae and algae can prolong the survival of Campylobacter species in co-culture

Several species of free-living amoebae can cause disease in humans. However, in addition to the direct pathogenicity of e.g. Acanthamoebae and Naegleria species, they are recognized as environmental hosts, indirectly involved in the epidemiology of many pathogenic bacteria. Although several studies have demonstrated intracellular survival of many different bacteria in these species, the extent of such interactions as well as the implications for the epidemiology of the bacterial species involved, are largely unknown and probably underestimated. In this study, we evaluated eight different unicellular eukaryotic organisms, for their potential to serve as environmental hosts for Campylobacter species. These organisms include four amoebozoas (Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba rhysodes and Hartmanella vermiformis), one alveolate (Tetrahymena pyriformis), one stramenopile (Dinobryon sertularia), one eugoenozoa (Euglena gracilis) and one heterolobosea (Naegleria americana). Campylobacter spp. including Campylobacter jejuni, Campylobacter coli and Campylobacter lari are the most common cause of gastroenteritis in the western world. Survival and replication of these three species as well as Campylobacter hyointestinalis were assessed in co-cultures with the eukaryotic organisms. Campylobacter spp. generally survived longer in co-cultures, compared to when incubated in the corresponding growth media. The eukaryotic species that best promoted bacterial survival was the golden algae D. sertularia. Three species of amoebozoas, of the genus Acanthamoeba promoted both prolonged survival and replication of Campylobacter spp. The high abundance in lakes, ponds and water distribution networks of these organisms indicate that they might have a role in the epidemiology of campylobacteriosis, possibly contributing to survival and dissemination of these intestinal pathogens to humans and other animals. The results suggest that not only C. jejuni, but a variety of Campylobacter spp. can interact with different eukaryotic unicellular organisms.     

Review of Campylobacter spp. in drinking and environmental waters

Consumption of contaminated drinking water is a significant cause of Campylobacter infections. Drinking water contamination is known to result from septic seepage and wastewater intrusion into non-disinfected sources of groundwater and occasionally from cross-connection into drinking water distribution systems. Wastewater effluents, farm animals and wild birds are the primary sources contributing human-infectious Campylobacters in environmental waters, impacting on recreational activities and drinking water sources. Culturing of Campylobacter entails time-consuming steps that often provide qualitative or semi-quantitative results. Viable but non-culturable forms due to environmental stress are not detected, and thus may result in false-negative assessments of Campylobacter risks from drinking and environmental waters. Molecular methods, especially quantitative PCR applications, are therefore important to use in the detection of environmental Campylobacter spp. Processing large volumes of water may be required to reach the desired sensitivity for either culture or molecular detection methods. In the future, applications of novel molecular techniques such as isothermal amplification and high-throughput sequencing applications are awaited to develop and become more affordable and practical in environmental Campylobacter research. The new technologies may change the knowledge on the prevalence and pathogenicity of the different Campylobacter species in the water environment.     

Optimization of Campylobacter growth conditions for further identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)

Growth conditions – including growth medium and incubation temperature – may influence the identification of Campylobacter by MALDI-TOF MS.     

Evaluation of propidium monoazide (PMA) treatment directly on membrane filter for the enumeration of viable but non cultivable Legionella by qPCR

A PMA (propidium monoazide) pretreatment protocol, in which PMA is applied directly to membrane filters, was developed for the PCR-based quantification (PMA-qPCR) of viable Legionella pneumophila. Using this method, the amplification of DNA from membrane-damaged L. pneumophila was strongly inhibited for samples containing a small number of dead bacteria.     

Isolation,identification and subtyping of Campylobacter: Where to from here?

Campylobacter species are widely regarded as the most frequent bacterial cause of gastroenteritis in humans worldwide. Their main transmission routes are via contaminated food and water. For interventions to be effective, methods for the detection, identification and epidemiological subtyping must be sensitive, accurate and rapid. As yet, methods are not perfect, although several significant advances have been made in these areas in recent years. This paper provides a brief review and commentary on the current state of the art in the hope that it will help provide context for others in selecting, improving or developing these vital tools for research and diagnoses.     

Review of current methodologies to isolate and identify Campylobacter spp. from foods

This article summarizes the most effective protocols to isolate Campylobacter spp. (mainly Campylobacter jejuni and Campylobacter coli) from food, primarily poultry products, and includes a summary of the current methods recommended by the Food and Drug Administration and the U.S. Department of Agriculture in the USA, and ISO in Europe. The recommended temperature for incubation of the samples throughout the isolation procedure is 42 °C. The enrichment of the samples for 48 h, which can be performed under aerobic conditions, is recommended to achieve a detectable number of Campylobacter cells. Bolton broth or buffered peptone water supplemented with cefoperazone and amphotericin B is commonly used enrichment broths. The transfer of the enriched samples to plate media using membrane filters helps to obtain pure Campylobacter colonies. Charcoal cefoperazone deoxycholate (CCDA) is the best choice among all plate media. There is no need to add oxygen quenching substances or blood to enrichment broth for the isolation of Campylobacter spp. However, the addition of blood to plate media aids in differential identification of presumptive colonies. Phase contrast microscopy and latex agglutination tests are confirmatory tests for presumptive Campylobacter isolates. The use of multiplex polymerase chain reaction (mPCR) assays is the simplest and most rapid method to identify isolates to the species level. mPCR assays, or other methods assessing DNA sequence variations, will probably become the confirmation procedure of choice in the future. Recent work with retail broiler meat has revealed that the rinsing of meat is more sensitive for the recovery of naturally contaminated retail broiler meat than current reference methods and requires less time for preparation and processing of the samples. This protocol could be coupled with DNA-based methods for a fast screening of positive samples.     

Quantification of viable Legionella pneumophila cells using propidium monoazide combined with quantitative PCR

One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50 μM PMA followed by 5 min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells.     

Campylobacter spp. detection in the 21st century: A review of the recent achievements in biosensor development

Campylobacter spp. are an important cause of acute bacterial diseases in humans worldwide. Many bacterial species in the Campylobacter genus are considered harmful and may cause several infectious diseases. Currently, there are no commercial biosensors available to detect Campylobacter spp. in food matrices, and little to no testing has been done in research laboratories with actual food matrices. Biosensors potentially provide a powerful means to detect Campylobacter spp. with the advantages of high sensitivity (low limits of detection with a high signal to noise ratio), high specificity (able to selectively detect the target among several similar targets), real time sensing, and in-site monitoring. This review summarizes the latest research in biosensing technologies for detection of Campylobacter spp. based on a variety of transducers and recognition elements. Finally, a comparison is made among all recently reported biosensors for the detection of Campylobacter spp.     

A small variation in diet influences the Lactobacillus strain composition in the crop of broiler chickens

Feed composition has the potential to influence the activities of bacteria that colonize the digestive tract of broiler chickens with important consequences for animal health, well being, and food safety. In this study, the gut microbiota of two groups of broiler chickens raised in immediate vicinity but fed either a standard corn/soybean meal ration (corn–soy, CS) or a ration high in wheat middlings (high wheat, HW) was characterized. The findings revealed that this small variation in feed composition did not influence the distribution of microbial species present in the microbial community throughout the digestive tract. However, diet variation markedly influenced the Lactobacillus strain composition in the crop. Most striking, the dominant type in birds on the CS diet (Lactobacillus agilis type R5), which comprised 25% of the isolates, was not detected in birds fed the HW diet. The latter birds harbored a different strain of L. agilis (type R1) in a significantly higher ratio than birds on the CS diet. Several other strains were also specific to the particular diet. In conclusion, this study showed that a small variation in the composition of chicken feed that does not result in detectable differences in species composition can still have an impact on which microbial strains become dominant in the digestive tract. This finding has relevance in the application of probiotics and other direct-fed microbials in poultry husbandry.     

Real-time PCR detection of Holophagae (Acidobacteria) and Verrucomicrobia subdivision 1 groups in bulk and leek (Allium porrum) rhizosphere soils

In the light of the poor culturability of Acidobacteria and Verrucomicrobia species, group-specific real-time (qPCR) systems were developed based on the 16S rRNA gene sequences from culturable representatives of both groups. The number of DNA targets from three different groups, i.e. Holophagae (Acidobacteria group 8) and Luteolibacter/Prosthecobacter and unclassified Verrucomicrobiaceae subdivision 1, was determined in DNA extracts from different leek (Allium porrum) rhizosphere soil compartments and from bulk soil with the aim to determine the distribution of the three bacterial groups in the plant-soil ecosystem. The specificity of the designed primers was evaluated in three steps. First, in silico tests were performed which demonstrated that all designed primers 100% matched with database sequences of their respective groups, whereas lower matches with other non-target bacterial groups were found. Second, PCR amplification with the different primer sets was performed on genomic DNA extracts from target and from non-target bacteria. This test demonstrated specificity of the designed primers for the target groups, as single amplicons of expected sizes were found only for the target bacteria. Third, the qPCR systems were tested for specific amplifications from soil DNA extracts and 48 amplicons from each primer system were sequenced. All sequences were > 97% similar to database sequences of the respective target groups. Estimated cell numbers based on Holophagae-, Luteolibacter/Prosthecobacter- and unclassified Verrucomicrobiaceae subdivision 1-specific qPCRs from leek rhizosphere compartments and bulk soils demonstrated higher preference for one or both rhizosphere compartments above bulk soil for all three bacterial groups.     

Improved protocol for isolation of Campylobacter spp. from retail broiler meat and use of pulsed field gel electrophoresis for the typing of isolates

To improve the detection of Campylobacter spp. in retail broiler meat, a reference method (R subsamples) based on the enrichment of 25 g of meat in Bolton broth at 42 °C under microaerobiosis was compared with an alternative method (A subsamples) consisting in the rinsing of meat samples for 30 s in buffered peptone water with antimicrobials with incubation at 42 °C under aerobiosis. One piece of meat (breasts, tenderloins and thighs) was rinse in experiment 1 (A1) and two pieces in experiment 2 (A2). Campylobacter spp. were isolated on agar plates and identified by PCR. Retail samples in Alabama had less prevalence (P ≤ 0.05) than samples in the state of Washington. The percentage of positive was higher (P ≤ 0.05) in A than in R subsamples and rinsing two pieces of meat yielded the highest percentage of positive subsamples. R subsamples showed variations in the prevalence by product. However, A subsamples had similar prevalence of positives among products compare to the result from reference method. More Campylobacter coli isolates were collected in A2 subsamples. Pulse field gel electrophoresis (PFGE) was used as subtyping method to study the genome similarity among the isolates from all methods. A larger diversity of isolates were detected by PFGE in A2 subsamples. Denaturing gradient gel electrophoresis analysis suggested that the initial bacterial populations of the meat samples impact the final bacterial profile after enrichment. Rinsing broiler meats was less time consuming, required less sample preparation and was more sensitive than the reference method for the isolation of naturally occurring Campylobacter spp. This new method could help with epidemiological and intervention studies to control Campylobacter spp.     

Real Time PCR to detect and differentiate Campylobacter fetus subspecies fetus and Campylobacter fetus subspecies venerealis

Bovine venereal campylobacter infection, caused by Campylobacter fetus venerealis, is of significant economic importance to the livestock industry. Unfortunately, the successful detection and discrimination of C. fetus venerealis from C. fetus fetus continue to be a limitation throughout the world. There are several publications warning of the problem with biotyping methods as well as with recent molecular based assays. In this study, assessed on 1071 isolates, we report on the successful development of two Real Time SYBR® Green PCR assays that will allow for the detection and discrimination of C. fetus fetus and C. fetus venerealis. The sensitivity reported here for the C. fetus (CampF4/R4) and the C. fetus venerealis (CampF7/R7) specific PCR assays are 100% and 98.7% respectively. The specificity for these same PCR assays are 99.6% and 99.8% respectively.     

Hedgehog does not guide migrating Drosophila germ cells

In many species, the germ cells, precursors of sperm and egg, migrate during embryogenesis. The signals that regulate this migration are thus essential for fertility. In flies, lipid signals have been shown to affect germ cell guidance. In particular, the synthesis of geranylgeranyl pyrophosphate through the 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (Hmgcr) pathway is critical for attracting germ cells to their target tissue. In a genetic analysis of signaling pathways known to affect cell migration of other migratory cells, we failed to find a role for the Hedgehog (Hh) pathway in germ cell migration. However, previous reports had implicated Hh as a germ cell attractant in flies and suggested that Hh signaling is enhanced through the action of the Hmgcr pathway. We therefore repeated several critical experiments and carried out further experiments to test specifically whether Hh is a germ cell attractant in flies. In contrast to previously reported findings and consistent with findings in zebrafish our data do not support the notion that Hh has a direct role in the guidance of migrating germ cells in flies.     

RNA interference of timeless gene does not disrupt circadian locomotor rhythms in the cricket Gryllus bimaculatus

Molecular studies revealed that autoregulatory negative feedback loops consisting of so-called “clock genes” constitute the circadian clock in Drosophila. However, this hypothesis is not fully supported in other insects and is thus to be examined. In the cricket Gryllus bimaculatus, we have previously shown that period (per) plays an essential role in the rhythm generation. In the present study, we cloned cDNA of the clock gene timeless (tim) and investigated its role in the cricket circadian oscillatory mechanism using RNA interference. Molecular structure of the cricket tim has rather high similarity to those of other insect species. Real-time RT-PCR analysis revealed that tim mRNA showed rhythmic expression in both LD and DD similar to that of per, peaking during the (subjective) night. When injected with tim double-stranded RNA (dstim), tim mRNA levels were significantly reduced and its circadian expression rhythm was eliminated. After the dstim treatment, however, adult crickets showed a clear locomotor rhythm in DD, with a free-running period significantly shorter than that of control crickets injected with Discosoma sp. Red2 (DsRed2) dsRNA. These results suggest that in the cricket, tim plays some role in fine-tuning of the free-running period but may not be essential for oscillation of the circadian clock.     

Phylogenic analysis of the genus Leishmania by cytochrome b gene sequencing

In a previous report (Luyo-Acero et al., 2004), we demonstrated that cytochrome b (Cyt b) gene analysis is an effective method for classifying several isolates of the genus Leishmania; hence, we have further applied this method to other Leishmania species in an effort to enhance the accuracy of the procedure and to construct a new phylogenic tree. In this study, a total of 30 Leishmania and Endotrypanum WHO reference strains, clinical isolates from our patients assigned to 28 strains (human and non-human pathogenic species) and two species of the genus Endotrypanum were analyzed. The Cyt b gene in each sample was amplified by PCR, and was then sequenced by several primers, as reported previously. The phylogenic tree was constructed based on the results obtained by the computer software MEGA v3.1 and PAUP* v4.0 Beta. The present phylogenic tree was almost identical to the traditional method of classification proposed by Lainson and Shaw (1987). However, it produces the following suggestions: (1) exclusion of L. (Leishmania) major from the L. (L.) tropica complex; (2) placement of L.tarentolae in the genus Sauroleishmania; (3) L. (L.) hertigi complex and L. (V.) equatorensis close to the genus Endotrypanum; (4) L. (L.) enrietti, defined as L. (L.) mexicana complex, placed in another position; and (5) L. (L.) turanica and L. (L.) arabica are located in an area far from human pathogenic Leishmania strains. Cyt b gene analysis is thus applicable to the analyzing phylogeny of the genus Leishmania and may be useful for separating non-human pathogenic species from human pathogenic species.     

Septal pore complex morphology in the Agaricomycotina (Basidiomycota) with emphasis on the Cantharellales and Hymenochaetales

The ultrastructure of septa and septum-associated septal pore caps are important taxonomic markers in the Agaricomycotina (Basidiomycota, Fungi). The septal pore caps covering the typical basidiomycetous dolipore septum are divided into three main phenotypically recognized morphotypes: vesicular-tubular (including the vesicular, sacculate, tubular, ampulliform, and globular morphotypes), imperforate, and perforate. Until recently, the septal pore cap-type reflected the higher-order relationships within the Agaricomycotina. However, the new classification of Fungi resulted in many changes including revision of existing and addition of new orders. Therefore, the septal pore cap ultrastructure of more than 325 species as reported in literature was related to this new classification. In addition, the septal pore cap ultrastructures of Rickenella fibula and Cantharellus formosus were examined by transmission electron microscopy. Both fungi have dolipore septa associated with perforate septal pore caps. These results combined with data from the literature show that the septal pore cap-type within orders of the Agaricomycotina is generally monomorphic, except for the Cantharellales and Hymenochaetales.It appears from the fungal phylogeny combined with the septal pore cap ultrastructure that the vesicular-tubular and the imperforate type both may have arisen from endoplasmic reticulum. Thereafter, the imperforate type eventually gave rise to the perforate septal pore cap-type.     

The influence of bioturbation by the sandprawn Callianassa kraussi on feeding and survival of the bivalve Eumarcia paupercula and the gastropod Nassarius kraussianus

Field observations and experimental evidence have shown that bioturbation by the southern African sandprawn Callianassa kraussi may significantly influence the abundance and distribution of the filter-feeding bivalve Eumarcia paupercula and the grazing gastropod Nassarius kraussianus. It was hypothesized that (1) sediment reworking by C. kraussi negatively affects microalgal growth on the sediment surface, leading to a reduction in food intake by N. kraussianus, (2) sediment deposited by C. kraussi will also diminish the food uptake of E. paupercula by interfering with its filtration mechanism. To test these hypotheses, manipulative field and laboratory experiments were undertaken in which N. kraussianus and E. paupercula were added to treatments with and without C. kraussi, and their survival and gut chlorophyll-a content measured. The effects of C. kraussi on sediment erodability and on condition of E. paupercula (tissue mass/shell length) were determined in a second experiment. In the presence of C. kraussi, (1) microalgal consumption by both N. kraussianus and E. paupercula was halved; (2) condition and survival of E. paupercula were significantly reduced but survival of N. kraussianus was unaffected; and (3) sediment erodability was increased. A significant negative relationship was established between sediment erodability and condition of E. paupercula. Evidently C. kraussi exerts a strongly negative influence on the feeding of E. paupercula and N. kraussianus, and this may explain the scarcity of these organisms in areas containing high densities of C. kraussi.