Clonal Population Structure and Antimicrobial Resistance of Campylobacter jejuni in Chicken Meat from Belgium

Campylobacter jejuni is one of the most important causes of human diarrhea worldwide. In the present work, multilocus sequence typing was used to study the genotypic diversity of 145 C. jejuni isolates from 135 chicken meat preparations sampled across Belgium. Isolates were further typed by pulsed-field gel electrophoresis, and their susceptibilities to six antimicrobials were determined. Fifty-seven sequence types (STs) were identified; 26.8% of the total typed isolates were ST-50, ST-45, or ST-257, belonging to clonal complex CC-21, CC-45, or CC-257, respectively. One clonal group comprised 22% (32/145) of all isolates, originating from five different companies and isolated over seven sampling months. Additionally, 53.1% of C. jejuni isolates were resistant to ciprofloxacin, and 48.2% were resistant to tetracycline; 28.9% (42/145) of all isolates were resistant to both ciprofloxacin and tetracycline. The correlation between certain C. jejuni clonal groups and resistance to ciprofloxacin and tetracycline was notable. C. jejuni isolates assigned to CC-21 (n = 35) were frequently resistant to ciprofloxacin (65.7%) and tetracycline (40%); however, 90% (18/20) of the isolates assigned to CC-45 were pansusceptible. The present study demonstrates that certain C. jejuni genotypes recur frequently in the chicken meat supply. The results of molecular typing, combined with data on sample sources, indicate a possible dissemination of C. jejuni clones with high resistance to ciprofloxacin and/or tetracycline. Whether certain clonal groups are common in the environment and repeatedly infect Belgian broiler flocks or whether they have the potential to persist on farms or in slaughterhouses needs further investigation.Campylobacter jejuni is among the most common bacterial causes of human gastroenteritis worldwide (4, 23). Infected humans exhibit a range of clinical symptoms from mild, watery diarrhea to severe inflammatory diarrhea (14). In addition, C. jejuni has been identified as an important infectious trigger for Guillain-Barré syndrome, the most common cause of acute flaccid paralysis in polio-free regions (16). Another issue of concern regarding Campylobacter is the increase in antimicrobial resistance appearing in various regions around the world (1). Infection with an antimicrobial-resistant Campylobacter strain may lead to a suboptimal outcome of antimicrobial treatment or even to treatment failure (11).Consumption of contaminated water and raw milk has been implicated in campylobacteriosis outbreaks (23). However, the majority of human cases are sporadic, and consumption or mishandling of contaminated raw or undercooked poultry meat is believed to be an important source of infection. Risk assessment studies, outbreak investigations, and case-control reports all incriminate chicken meat as a major source, perhaps the major source, of food-borne transmission (14, 17, 32, 48). In Belgium in 1999, a controlled withdrawal of poultry products from sale due to alleged dioxin contamination resulted in a 40% reduction in the frequency of human campylobacteriosis (44). Thereafter and since the year 2000, the Campylobacter contamination of Belgian poultry carcasses and meat has been monitored by the Federal Agency for the Safety of the Food Chain, and the rate of positive samples is regarded as high. In 2006, 55.5% of cecal samples (n = 6,443) from Belgian broilers at slaughter tested positive for Campylobacter (3). In 2007, an industry-focused survey reported that 48% of Belgian chicken meat preparations (n = 656) were contaminated with Campylobacter (19).Molecular typing is an important tool in elucidating the diversity and transmission routes of Campylobacter isolates contaminating the food chain. In the United States, molecular analysis of Campylobacter spp. from poultry production and processing environments showed that many of the clones found within a flock are present in the final products, although the diversity of Campylobacter isolates in the final product was lower than that observed in the flock (22). Furthermore, numerous molecular epidemiological studies indicate that the genotypes of C. jejuni isolated from human cases overlap those of poultry origin (17, 47). Various molecular typing methods for the study of the population structure of Campylobacter are currently available (46). Among these, the multilocus sequence typing (MLST) approach is an emerging tool for research on the population structure and molecular epidemiology of Campylobacter. The technique is highly reproducible, portable, and easy to interpret, and results can be shared through a publicly accessible online database (31, 34). As such, MLST is becoming an important tool for studying the molecular epidemiology of Campylobacter in a global context. The accumulation of sequence typing data generated from different countries and settings could allow the creation of more-sophisticated models of the epidemiology and evolution of bacterial pathogens and the development of improved approaches for combating their spread (41).In Belgium, there is a paucity of information regarding the population structure of Campylobacter in the chicken meat supply. No population-based surveys have been conducted to investigate the molecular epidemiology of C. jejuni in chicken meat at points close to human consumption. In this study, MLST and pulsed-field gel electrophoresis (PFGE) were used to characterize the diversity of, and clonal relationships among, 145 C. jejuni isolates from Belgian chicken meat preparations. In addition, we characterized the antimicrobial resistance in this collection and correlated it with C. jejuni genotypes.     

Hydrophobic Surface Patches on LolA of Pseudomonas aeruginosa Are Essential for Lipoprotein Binding

Many lipoproteins reside in the outer membrane (OM) of Gram-negative bacteria, and their biogenesis is dependent on the Lol (localization of lipoproteins) system. The periplasmic chaperone LolA accepts OM-destined lipoproteins that are released from the inner membrane by the LolCDE complex and transfers them to the OM receptor LolB. The exact nature of the LolA-lipoprotein complex is still unknown. The crystal structure of Escherichia coli LolA features an open β-barrel covered by α helices that together constitute a hydrophobic cavity, which would allow the binding of one acyl chain. However, OM lipoproteins contain three acyl chains, and the stoichiometry of the LolA-lipoprotein complex is 1:1. Here we present the crystal structure of Pseudomonas aeruginosa LolA that projects clear hydrophobic surface patches. Since these patches are large enough to accommodate acyl chains, their role in lipoprotein binding was investigated. Several LolA mutant proteins were created, and their functionality was assessed by studying their capacity to release lipoproteins produced in sphaeroplasts. Interruption of the largest hydrophobic patch completely destroyed the lipoprotein-releasing capacity of LolA, while interruption of smaller patches apparently reduced efficiency. Thus, the results show a new lipoprotein transport model that places (some of) the acyl chains on the hydrophobic surface patches.     

The incidence of nirS and nirK and their genetic heterogeneity in cultivated denitrifiers

Gene sequence analysis of nirS and nirK, both encoding nitrite reductases, was performed on cultivated denitrifiers to assess their incidence in different bacterial taxa and their taxonomical value. Almost half of the 227 investigated denitrifying strains did not render an nir amplicon with any of five previously described primers. NirK and nirS were found to be prevalent in Alphaproteobacteria and Betaproteobacteria, respectively, nirK was detected in the Firmicutes and Bacteroidetes and nirS and nirK with equal frequency in the Gammaproteobacteria. These observations deviated from the hitherto reported incidence of nir genes in bacterial taxa. NirS gene phylogeny was congruent with the 16S rRNA gene phylogeny on family or genus level, although some strains did group within clusters of other bacterial classes. Phylogenetic nirK gene sequence analysis was incongruent with the 16S rRNA gene phylogeny. NirK sequences were also found to be significantly more similar to nirK sequences from the same habitat than to nirK sequences retrieved from highly related taxa. This study supports the hypothesis that horizontal gene transfer events of denitrification genes have occurred and underlines that denitrification genes should not be linked with organism diversity of denitrifiers in cultivation-independent studies.     

Arabidopsis PASTICCINO2 is an antiphosphatase involved in regulation of cyclin-dependent kinase A

PASTICCINO2 (PAS2), a member of the protein Tyr phosphatase-like family, is conserved among all eukaryotes and is characterized by a mutated catalytic site. The cellular functions of the Tyr phosphatase-like proteins are still unknown, even if they are essential in yeast and mammals. Here, we demonstrate that PAS2 interacts with a cyclin-dependent kinase (CDK) that is phosphorylated on Tyr and not with its unphosphorylated isoform. Phosphorylation of the conserved regulatory Tyr-15 is involved in the binding of CDK to PAS2. Loss of the PAS2 function dephosphorylated Arabidopsis thaliana CDKA;1 and upregulated its kinase activity. In accordance with its role as a negative regulator of the cell cycle, overexpression of PAS2 slowed down cell division in suspension cell cultures at the G2-to-M transition and early mitosis and inhibited Arabidopsis seedling growth. The latter was accompanied by altered leaf development and accelerated cotyledon senescence. PAS2 was localized in the cytoplasm of dividing cells but moved into the nucleus upon cell differentiation, suggesting that the balance between cell division and differentiation is regulated through the interaction between CDKA;1 and the antiphosphatase PAS2.     

Crystal structure of Pterocarpus angolensis lectin in complex with glucose,sucrose, and turanose

The crystal structure of the Man/Glc-specific seed lectin from Pterocarpus angolensis was determined in complex with methyl-alpha-d-glucose, sucrose, and turanose. The carbohydrate binding site contains a classic Man/Glc type specificity loop. Its metal binding loop on the other hand is of the long type, different from what is observed in other Man/Glc-specific legume lectins. Glucose binding in the primary binding site is reminiscent of the glucose complexes of concanavalin A and lentil lectin. Sucrose is found to be bound in a conformation similar as seen in the binding site of lentil lectin. A direct hydrogen bond between Ser-137(OG) to Fru(O2) in Pterocarpus angolensis lectin replaces a water-mediated interaction in the equivalent complex of lentil lectin. In the turanose complex, the binding site of the first molecule in the asymmetric unit contains the alphaGlc1-3betaFruf form of furanose while the second molecule contains the alphaGlc1-3betaFrup form in its binding site.     

Secondary-structure characterization of two proficient kinase deoxyribozymes

Dk1 and Dk2 are two catalytically proficient, manganese-dependent, guanine-rich deoxyribozymes previously isolated for DNA phosphorylation. In this study, we carried out a series of experiments that aimed to understand the structural properties of Dk1 and Dk2 and compare the structural similarities or differences of these two distinct deoxyribozymes that carry out similar catalytic functions. First, we performed reselections from two partially randomized DNA libraries on the basis of the original Dk1 and Dk2 sequences to isolate catalytically active sequence variants and identify nucleotides that are invariable, well-conserved, or highly mutagenized. Sequence analysis of these variants assisted by secondary-structure predictions led to the identification of possible Watson-Crick base-pairing regions within each deoxyribozyme. Sequence truncation and base-pair partner exchange experiments were conducted to confirm, or rule out, the existence of the predicted secondary-structure elements. Finally, methylation interference experiments were applied to identify nucleotides that are potentially important for the tertiary structure folding of the deoxyribozymes. Our data suggest that Dk1 and Dk2, despite the differences in their primary sequences and NTP requirements, use an analogous stem-loop element to anchor a structural domain of substantial tertiary interactions to execute their catalytic functions.