Comparative Genomic Analysis Identifies a Campylobacter Clade Deficient in Selenium Metabolism.

The nonthermotolerant Campylobacter species C. fetus, C. hyointestinalis, C. iguaniorum, and C. lanienae form a distinct phylogenetic cluster within the genus. These species are primarily isolated from foraging (swine) or grazing (e.g., cattle, sheep) animals and cause sporadic and infrequent human illness. Previous typing studies identified three putative novel C. lanienae-related taxa, based on either MLST or atpA sequence data. To further characterize these putative novel taxa and the C. fetus group as a whole, 76 genomes were sequenced, either to completion or to draft level. These genomes represent 26 C. lanienae strains and 50 strains of the three novel taxa. C. fetus, C. hyointestinalis and C. iguaniorum genomes were previously sequenced to completion; therefore, a comparative genomic analysis across the entire C. fetus group was conducted (including average nucleotide identity analysis) that supports the initial identification of these three novel Campylobacter species. Furthermore, C. lanienae and the three putative novel species form a discrete clade within the C. fetus group, which we have termed the C. lanienae clade. This clade is distinguished from other members of the C. fetus group by a reduced genome size and distinct CRISPR/Cas systems. Moreover, there are two signature characteristics of the C. lanienae clade. C. lanienae clade genomes carry four to ten unlinked and similar, but nonidentical, flagellin genes. Additionally, all 76 C. lanienae clade genomes sequenced demonstrate a complete absence of genes related to selenium metabolism, including genes encoding the selenocysteine insertion machinery, selenoproteins, and the selenocysteinyl tRNA.

Cardamom, Cumin, and Dill Weed Essential Oils: Chemical Compositions, Antimicrobial Activities, and Mechanisms of Action against Campylobacter spp.

Natural antimicrobials as well as essential oils (EOs) have gained interest to inhibit pathogenic microorganisms and to control food borne diseases. Campylobacter spp. are one of the most common causative agents of gastroenteritis. In this study, cardamom, cumin, and dill weed EOs were evaluated for their antibacterial activities against Campylobacter jejuni and Campylobacter coli by using agar-well diffusion and broth microdilution methods, along with the mechanisms of antimicrobial action. Chemical compositions of EOs were also tested by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The results showed that cardamom and dill weed EOs possess greater antimicrobial activity than cumin with larger inhibition zones and lower minimum inhibitory concentrations. The permeability of cell membrane and cell membrane integrity were evaluated by determining relative electric conductivity and release of cell constituents into supernatant at 260 nm, respectively. Moreover, effect of EOs on the cell membrane of Campylobacter spp. was also investigated by measuring extracellular ATP concentration. Increase of relative electric conductivity, extracellular ATP concentration, and cell constituents' release after treatment with EOs demonstrated that tested EOs affected the membrane integrity of Campylobacter spp. The results supported high efficiency of cardamom, cumin, and dill weed EOs to inhibit Campylobacter spp. by impairing the bacterial cell membrane.

A modified bioautographic method for antibacterial component screening against anaerobic and microaerophilic bacteria.

Direct bioautography is a useful method to identify antimicrobial compounds with potential therapeutic importance. Because of technical limitations till now, it has been applied only for aerobic bacteria. In this work we present the modification of the original method by which antimicrobial screening of bacteria requiring modified atmosphere became feasible by direct bioautography. Here we demonstrate its applicability by testing three anaerobic Clostridium perfringens and three microaerophilic Campylobacter jejuni strains against two essential oils, clove and thyme. Antimicrobial component profiles of clove and thyme essential oils against these two medically important pathogenic bacteria were compared and significant differences were revealed in their inhibition capacities. Linalool, a component of thyme essential oil exerted a more expressed antibacterial activity against C. perfringens than against C. jejuni. Our results demonstrate that direct bioautography is not only suitable for testing aerobic bacteria, but by applying the presently described modified version it can also contribute to the quest to find novel antimicrobial agents against multidrug resistant anaerobic and microaerophilic bacteria.

Modification of Karmali agar by supplementation with potassium clavulanate for the isolation of Campylobacter from chicken carcass rinses.

The detection ability and selectivity of Karmali agar was improved by supplementation of an extended-spectrum ß-lactamase inhibitor, potassium clavulanate. The optimum concentration of potassium clavulanate (0.5 µg/ml) in Karmali agar was determined by inoculation of 50 Campylobacter and 30 extended-spectrum ß-lactamase-producing E. coli strains onto normal and modified Karmali agar containing various concentrations of the agent. Eighty retail carcasses were rinsed with 400 ml of buffered peptone water. The rinse samples were enriched in 2 × blood-free Bolton enrichment broth at 42°C for 48 h and then were streaked onto normal and modified Karmali agar containing 0.5 µg/ml potassium clavulanate. The suspicious colonies were subcultured on Columbia blood agar and confirmed by colony PCR. In chicken carcass samples, the modified Karmali agar showed a significantly greater isolation rate than normal Karmali agar (42.5 versus 21.3%; P < 0.05). Furthermore, the selectivity of the modified Karmali agar was also significantly higher (P < 0.05) than that of the normal Karmali agar, as seen by comparison of the number of contaminated agar plates (83.8 versus 97.5%) and the growth index (1.67 versus 2.91) of the non-Campylobacter colonies.

Specific TonB-ExbB-ExbD energy transduction systems required for ferric enterobactin acquisition in Campylobacter.

Ferric enterobactin (FeEnt) acquisition plays a critical role in the pathophysiology of Campylobacter, the leading bacterial cause of human gastroenteritis in industrialized countries. In Campylobacter, the surface-exposed receptor, CfrA or CfrB, functions as a 'gatekeeper' for initial binding of FeEnt. Subsequent transport across the outer membrane is energized by TonB-ExbB-ExbD energy transduction systems. Although there are up to three TonB-ExbB-ExbD systems in Campylobacter, the cognate components of TonB-ExbB-ExbD for FeEnt acquisition are still largely unknown. In this study, we addressed this issue using complementary molecular approaches: comparative genomic analysis, random transposon mutagenesis and site-directed mutagenesis in two representative C. jejuni strains, NCTC 11168 and 81-176. We demonstrated that CfrB could interact with either TonB2 or TonB3 for efficient Ent-mediated iron acquisition. However, TonB3 is a dominant player in the CfrA-dependent pathway. The ExbB2 and ExbD2 components were essential for both CfrA- and CfrB-dependent FeEnt acquisition. Sequences analysis identified potential TonB boxes in CfrA and CfrB, and the corresponding binding sites in TonB. In conclusion, these findings identify specific TonB-ExbB-ExbD energy transduction components required for FeEnt acquisition, and provide insights into the complex molecular interactions of FeEnt acquisition systems in Campylobacter.

Prevalence, haemolytic and haemagglutination activities and antibiotic susceptibility profiles of Campylobacter spp. isolated from human diarrhoeal stools in Vhembe District, South Africa.

Campylobacter species are increasingly being recognized as agents of gastroenteritis worldwide. However, data on the pathogenic characteristics of the organism isolated in rural communities in South Africa are lacking. In this study, the prevalence of Campylobacter spp. from diarrhoeal stools, haemolytic and haemagglutinating activities of the isolates, and antibiotic susceptibility profiles, including minimum inhibitory concentration (MIC) patterns to different antibiotics, were determined using the standard microbiological techniques. Campylobacter spp. were isolated from individuals of all age-groups; however, the infection rate was higher among individuals aged less than two years (30.4%). Of 115 Campylobacter strains isolated, polymerase chain reaction (PCR) analysis indicated that 98 (85%) were C. jejuni, while 17 (15%) were C. coli. Seventy-one (62%) of the strains showed haemolysis on human blood, and 80% agglutinated human blood, whereas 22.6% were beta-lactamase-positive. Resistance to antimicrobials, such as erythromycin, ciprofloxacin, vancomycin, and fusidic acid, was high. Increased resistance to macrolide and quinolone antibiotics poses major risks for treatment failure. Haemolytic and haemagglutinating activities may be useful in preliminary characterization of pathogenic strains in settings where Campylobacter-associated infections are common.

Influence of media supplements on growth and survival of Campylobacter pylori.

Experiments were designed to determine the role of heme and the importance of other factors in the growth of Campylobacter pylori. Campylobacter pylori strains were tested for their ability to synthesize porphyrin, for their ability to grow and be maintained on basal medium and basal medium supplemented with blood or blood products, and for the influence of bovine serum albumin and catalase on viability. Results indicated that Campylobacter pylori does not require heme as a source of porphyrin. Growth of Campylobacter pylori could not be sustained on media containing starch or hemoglobin, but was sustained on media containing erythrocytes, serum, bovine serum albumin or catalase. The ability to grow on media containing bovine serum albumin and catalase suggests that protection from toxic fatty acids and the prevention of toxic product formation may be important factors in the growth and survival of Campylobacter pylori in vitro. Both bovine serum albumin and catalase combined provide the minimum requirements which allow the spectrum of Campylobacter pylori present in a single culture to grow on blood-free media.