Histo-blood group antigens as allo- and autoantigens

The science of blood groups has made giant steps forward during the last decade. Blood-group typing of red blood cells (RBCs) is performed on more than 15 million samples per year in Europe, today much less often for forensic reasons than for clinical purposes such as transfusion and organ transplantation. Specific monoclonal antibodies are used with interpretation on the basis of RBC agglutination patterns, and mass genotyping may well be on its way to becoming a routine procedure. The discovery that most blood group systems, whose antigens are by definition found on RBCs, are also expressed in multiple other tissues has sparked the interest of transplantation medicine in immunohematology beyond the HLA system. The one and only "histo-blood group" (HBG) system that is routinely considered in transplantation medicine is ABO, because ABO antigen-incompatible donor/recipient constellations are preferably avoided. However, other HBG systems may also play a role, thus far underestimated. This paper is an up-to-date analysis of the importance of HBG systems in the alloimmunity of transplantation and autoimmune events, such as hemolytic anemia.

Solid organ transplantation across the ABO histo-blood group barrier: a case report

The ABO blood group system until recently constituted an insuperable barrier for solid organ transplantation, but cases of heart transplantation in infants and kidney transplantation in adults have been reported, wherein ABO-incompatible grafts have been successful. In 1990, the molecular genetic basis of three major alleles at the ABO locus was elucidated; A and B glycosyltransferases are specified by a variety of functional alleles at this locus. The antibody response to ABH antigens, namely, naturally occurring anti-A/B IgM and IgG isotype agglutinins, are controlled preoperatively by recipient conditioning using plasma exchange, immunoadsorption, and immunosuppressive regimens. We report an O-type patient who accidentally received a B-type cardiac allograft in 1997 who survived for 5 years, dying for an unrelated reason. Over a period of 45 months semiquantitatively we monitored the expression of ABO-type antigens in graft heart vessels using monoclonal antibodies on sections of formalin-fixed, paraffin-embedded biopsies. We observed a progressive change in the antigenic profile of graft endothelial cells from B- to O-type, which was first detected at 1 year posttransplant and most prominent 3 years later, the end of the observation period. No temporal relationship was observed between the transition from B to O expression, the anti-B antibody levels or the immunosuppressive regimen.

Outer membrane porin M35 of Moraxella catarrhalis mediates susceptibility to aminopenicillins

BACKGROUND: The outer membrane protein M35 is a conserved porin of type 1 strains of the respiratory pathogen Moraxella catarrhalis. It was previously shown that M35 is involved in the uptake of essential nutrients required for bacterial growth and for nasal colonization in mice. The aim of this study was (i) to characterize the potential roles of M35 in the host-pathogen interactions considering the known multifunctionality of porins and (ii) to characterize the degree of conservation in the phylogenetic older subpopulation (type 2) of M. catarrhalis. RESULTS: Isogenic m35 mutants of the type 1 strains O35E, 300 and 415 were tested for their antimicrobial susceptibility against 15 different agents. Differences in the MIC (Minimum Inhibitory Concentration) between wild-type and mutant strains were found for eight antibiotics. For ampicillin and amoxicillin, we observed a statistically significant 2.5 to 2.9-fold MIC increase (p < 0.03) in the m35 mutants. Immunoblot analysis demonstrated that human saliva contains anti-M35 IgA. Wild-type strains and their respective m35 mutants were indistinguishable with respect to the phenotypes of autoagglutination, serum resistance, iron acquisition from human lactoferrin, adherence to and invasion of respiratory tract epithelial cells, and proinflammatory stimulation of human monocytes. DNA sequencing of m35 from the phylogenetic subpopulation type 2 strain 287 revealed 94.2% and 92.8% identity on the DNA and amino acid levels, respectively, in comparison with type 1 strains. CONCLUSION: The increase in MIC for ampicillin and amoxicillin, respectively, in the M35-deficient mutants indicates that this porin affects the outer membrane permeability for aminopenicillins in a clinically relevant manner. The presence of IgA antibodies in healthy human donors indicates that M35 is expressed in vivo and recognized as a mucosal antigen by the human host. However, immunoblot analysis of human saliva suggests the possibility of antigenic variation of immunoreactive epitopes, which warrants further analysis before M35 can be considered a potential vaccine candidate.

Effects of HIV type-1 immune selection on susceptability to integrase inhibitor resistance

BACKGROUND: All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1 integrase sequences from 342 antiretroviral-naive individuals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. METHODS: Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8(+) T-cell escape mutations. RESULTS: In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. CONCLUSIONS: Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.

Classification of the taxon 2 and taxon 3 complex of Bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp. nov., Gallibacterium trehalosifermentans sp. nov. and Gallibacterium salpingitidis sp. nov

This investigation was based on 23 isolates from several European countries collected over the past 30 years, and included characterization of all isolates. Published data on amplified fragment length polymorphism typing of isolates representing all biovars as well as protein profiles were used to select strains that were then further characterized by polyamine profiling and sequencing of 16S rRNA, infB, rpoB and recN genes. Comparison of 16S rRNA gene sequences revealed a monophyletic group within the avian 16S rRNA group of the Pasteurellaceae, which currently includes the genera Avibacterium, Gallibacterium and Volucribacter. Five monophyletic subgroups related to Gallibacterium anatis were recognized by 16S rRNA, rpoB, infB and recN gene sequence comparisons. Whole-genome similarity between strains of the five subgroups and the type strain of G. anatis calculated from recN sequences allowed us to classify them within the genus Gallibacterium. In addition, phenotypic data including biochemical traits, protein profiling and polyamine patterns clearly indicated that these taxa are related. Major phenotypic diversity was observed for 16S rRNA gene sequence groups. Furthermore, comparison of whole-genome similarities, phenotypic data and published data on amplified fragment length polymorphism and protein profiling revealed that each of the five groups present unique properties that allow the proposal of three novel species of Gallibacterium, for which we propose the names Gallibacterium melopsittaci sp. nov. (type strain F450(T) =CCUG 36331(T) =CCM 7538(T)), Gallibacterium trehalosifermentans sp. nov. (type strain 52/S3/90(T) =CCUG 55631(T) =CCM 7539(T)) and Gallibacterium salpingitidis sp. nov. (type strain F150(T) =CCUG 15564(T) =CCUG 36325(T) =NCTC 11414(T)), a novel genomospecies 3 of Gallibacterium and an unnamed taxon (group V). An emended description of the genus Gallibacterium is also presented.

Phenotypic and genetic characterization of Pasteurella multocida and related isolates from rabbits in Switzerland

Several bacteria belonging to the family Pasteurellaceae are potential pathogens in rabbits. In particular, Pasteurella multocida is considered to be important, and outbreaks caused by this species result in considerable economic losses in rabbitries. However, Pasteurellaceae spp. isolated from rabbits are poorly characterized, and thus, proper identification of P. multocida isolates from these animals is problematic and often unsatisfactory, thereby hampering epidemiological investigations. Therefore, 228 isolates from rabbit populations originating from a breeding and fattening organization with group management and postmortem cases with pasteurellosis from individual owners were phenotypically and genotypically analyzed using biochemical tests and repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Furthermore, 41 samples representing observed phenotypes were selected for phylogenetic analysis using 16S ribosomal RNA and rpoB genes. The REP-PCR typing and phylogenetic analyses correlated well and appeared to be distinct molecular methods for characterization of rabbit isolates. Phenotyping, however, diverged from molecular recognition, reflecting the problematic conventional diagnosis of these strains. The fermentation of sorbitol appeared to be an imprecise indicator for P. multocida subspecies classification. According to REP-PCR and sequencing results, 82% of the isolates were characterized as P. multocida subsp. multocida, 3% as P. multocida subsp. septica, and 5% as P. multocida. Further, 5% were identified as Pasteurella canis. The other 5% represented a homogeneous group of unknown species belonging to the Pasteurellaceae. Samples obtained from individual postmortem cases demonstrated a higher phenotypic and genetic heterogeneity than samples from group management rabbits.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Staphylococcus pseudintermedius in the dog: taxonomy, diagnostics, ecology, epidemiology and pathogenicity

The dog is the natural host of Staphylococcus pseudintermedius. Many research efforts are currently being undertaken to expand our knowledge and understanding of this important canine commensal and opportunistic pathogen. The objective of this review is to summarize the current knowledge of the species, including the latest research outcomes, with emphasis on taxonomy, diagnostics, ecology, epidemiology and pathogenicity. Despite the important taxonomic changes that have occurred over the past few years, the risk of misidentification in canine specimens is low and does not have serious consequences for clinical practice. Staphylococcus pseudintermedius carriage in the dog is more frequent and genetically heterogeneous compared with that of Staphylococcus aureus in man. It appears that these staphylococcal species have evolved separately through adaptation to their respective natural hosts and differ with regard to various aspects concerning ecology, population structure and evolution of antibiotic resistance. Further understanding of the ecology and epidemiology of S. pseudintermedius is hampered by the lack of a standard method for rapid and discriminatory typing and by the limited data available on longitudinal carriage and population structure of meticillin-susceptible strains. With regard to pathogenicity, it is only now that we are starting to explore the virulence potential of S. pseudintermedius based on genomic and proteomic approaches, and more research is needed to assess the importance of individual virulence factors and the possible existence of hypervirulent strains.

Genotypes and antibiotic resistance of Campylobacter coli in fattening pigs

Campylobacter coli is a food-borne zoonotic pathogen causing human gastroenteritis worldwide. The organism is a commensal in the intestine of many food production animals including fattening pigs. The role of the pig as a potential reservoir for C. coli affecting human either directly or via poultry has hardly been investigated and genetic characterization of porcine strains is needed to address this question. For this aim multilocus sequence typing (MLST) and flaB typing was applied to 256 C. coli isolates from faeces of fattening pig collected during 2009 at different slaughterhouses in Switzerland. In addition genotypic resistances towards macrolides and quinolones based on point mutations in the 23S rRNA and gyrA genes, respectively, were determined. Of the 67 sequence types (STs) obtained by MLST, 37 were found for the first time. flaB typing revealed 46 different types with 14 of them being novel and was useful to further differentiate strains with an identical ST. Quinolone resistance was detected in 33.6% and macrolide resistance was found in 10.6% of isolates. Comparison with 99 C. coli pig isolates from 2001 revealed a significant decrease in antibiotic resistance towards both groups of antibiotics and there was high overlap between genotypes of 2001 and 2009. Little overlap of porcine genotypes was found with 97 C. coli isolates from poultry collected 2008, however, macrolide resistance was significantly higher in pig isolates. In conclusion, C. coli from Swiss pig are heterogeneous containing many novel STs, findings that could reflect the partitioned Swiss pig production with almost no international breed exchange. The antibiotic resistance echoes the use of corresponding drugs in the Swiss livestock production and indicates the efficacy of restrictive application of antibiotics in order to reduce resistances.

Comparison of genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli on chicken retail meat and at slaughter

Multilocus sequence typing (MLST) and antibiotic resistance patterns of Campylobacter jejuni and Campylobacter coli from retail chicken meat showed high overlap with isolates collected at slaughterhouses, indicating little selection along the production chain. They also showed significant common sequence types with human clinical isolates, revealing chicken meat as a likely source for human infection.

Genotypes and antibiotic resistances of Campylobacter jejuni and Campylobacter coli isolates from domestic and travel-associated human cases

Multilocus sequence typing (MLST) extended with flaB typing of 425 Campylobacter jejuni isolates and 42 Campylobacter coli isolates revealed quite a low overlap between human isolates from travel-associated and domestic cases in Switzerland. Men were more frequently affected by Campylobacter than women, but strains from women and, overall, from travel-associated cases showed mutations conferring quinolone resistance more frequently than strains from men and domestic cases, respectively.

Mesenteric lymphangitis and sepsis due to RTX toxin-producing Actinobacillus spp in 2 foals with hypothyroidism-dysmaturity syndrome

Actinobacillus suis-like organisms (ASLOs) have been isolated from the genital, respiratory, and digestive tracts of healthy adult horses, horses with respiratory disease, and septic foals. Two foals with congenital hypothyroidism-dysmaturity syndrome from separate farms developed ASLO infection. At necropsy, both had contracted carpal flexor tendons, thyroid hyperplasia, and thrombotic and necrotizing mesenteric lymphangitis and lymphadenitis; one foal also had mandibular prognathism. Numerous ASLOs were isolated from tissues from both foals, including intestine. Biochemical testing and mass spectrometric analysis of the two Actinobacillus isolates did not allow unequivocal identification. Comparative genetic analysis was done on these and similar isolates, including phylogeny based on 16S rRNA, rpoB and recN genes, as well as RTX (repeat in toxin) toxin typing of apxIA-apxIVA and aqxA genes. One isolate was identified as Actinobacillus suis sensu stricto, based on the presence of apxIA and apxIIA but not aqxA, whereas the other isolate had aqxA but neither apxIA nor apxIIA, consistent with A equuli ssp haemolyticus. Based on genotypic analysis of the isolates included for comparison, 3 of 3 equine ASLOs and 2 of 5 A equuli isolates were reclassified as A equuli subsp haemolyticus, emphasizing the importance of toxin genotyping in accurate classification of actinobacilli.

Molecular identification and epidemiological tracing of Pasteurella multocida meningitis in a baby

We report a case of Pasteurella multocida meningitis in a 1-month-old baby exposed to close contact with two dogs and a cat but without any known history of injury by these animals. 16S rRNA gene sequencing of the isolate from the baby allowed identification at the subspecies level and pointed to the cat as a possible source of infection. Molecular typing of Pasteurella isolates from the animals, from the baby, and from unrelated animals clearly confirmed that the cat harbored the same P. multocida subsp. septica strain on its tonsils as the one isolated from the cerebrospinal fluid of the baby. This case stresses the necessity of informing susceptible hosts at risk of contracting zoonotic agents about some basic hygiene rules when keeping pets. In addition, this study illustrates the usefulness of molecular methods for identification and epidemiological tracing of Pasteurella isolates.

Genotypes and antibiotic resistance of canine Campylobacter jejuni isolates

Campylobacter jejuni is the most important cause of bacterial gastroenteritis in humans. It is a commensal in many wild and domestic animals, including dogs. Whereas genotypes of human and chicken C. jejuni isolates have been described in some detail, only little information on canine C. jejuni genotypes is available. To gain more information on genotypes of canine C. jejuni and their zoonotic potential, isolates from routine diagnostics of diarrheic dogs as well as isolates of a prevalence study in non-diarrheic dogs were analyzed. Prevalence of thermophilic Campylobacter among non-diarrheic dogs was 6.3% for C. jejuni, 5.9% for Campylobacter upsaliensis and 0.7% for Campylobacter coli. The C. jejuni isolates were genotyped by multi locus sequence typing (MLST) and flaB typing. Resistance to macrolides and quinolones was genetically determined in parallel. Within the 134 genotyped C. jejuni isolates 57 different sequence types (ST) were found. Five STs were previously unrecognized. The most common STs were ST-48 (11.2%), ST-45 (10.5%) and ST-21 (6.0%). Whereas no macrolide resistance was found, 28 isolates (20.9%) were resistant to quinolones. ST-45 was significantly more prevalent in diarrheic than in non-diarrheic dogs. Within the common time frame of isolation 94% of the canine isolates had a ST that was also found in human clinical isolates. In conclusion, prevalence of C. jejuni in Swiss dogs is low but there is a large genetic overlap between dog and human isolates. Given the close contact between human and dogs, the latter should not be ignored as a potential source of human campylobacteriosis.

Copy number variation of the Beta-defensin genes in europeans: no supporting evidence for association with lung function, chronic obstructive pulmonary disease or asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02-1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72-1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.