Soft Tissue Infection of Immunocompetent Man with Cat-Derived Globicatella Species.

We report a novel Globicatella species causing extensive soft tissue infection in a man bitten by a stray domestic cat in the United Kingdom. We identified this bacterium by 16S rRNA gene sequencing, whole-genome sequencing, and biochemical profiling and determined antimicrobial drug susceptibility.

Bartonella clarridgeiae infection in a patient with aortic root abscess and endocarditis.

INTRODUCTION: Bartonella species are increasingly recognized as agents of culture-negative endocarditis. However, to date, almost all human cases have been associated with two members of the genus, Bartonella henselae and Bartonella quintana. B. henselae infections are zoonotic, with domestic cats serving as reservoir hosts for the pathogen. Bartonella clarridgeiae also exploits cats as reservoir hosts, but its zoonotic potential is far less established. CASE PRESENTATION: A 34-year-old male presented with palpitations after a history of aortic incompetence. During surgery for an aortic valve replacement, two vegetations were found on the aortic valve. PCR analysis of the vegetation demonstrated the presence of Bartonella species and so the patient was treated post-operatively with ceftriaxone and doxycycline, making a good recovery. Further PCR-based analysis of the patient's aortic vegetation confirmed the presence of B. clarridgeiae . CONCLUSION: This report expands the number of Bartonella species associated with endocarditis and provides clear evidence that B. clarridgeiae should be considered a zoonotic pathogen.

rpoB gene sequencing highlights the prevalence of an E. miricola cluster over other Elizabethkingia species among UK cystic fibrosis patients.

Difficulties in distinguishing species of the Elizabethkingia genus by MALDI-TOF prompted use of rpoB sequencing to investigate species distribution among 44 isolates from cystic fibrosis (CF) patients. Forty-three isolates from 38 patients formed a cluster comprising E. miricola and proposed novel species E. bruuniana sp. nov., the exception clustering with proposed species E. ursingii sp. nov., also part of this wider cluster. All 44 isolates were PCR-positive for urease gene ureG, whereas only one of 23 E. anophelis isolates from non-CF patients was positive, suggesting that this gene is largely associated with the E. miricola cluster. Antibiotic susceptibilities of 12 CF isolates revealed all were resistant to beta-lactams with the exception of piperacillin-tazobactam, and were only susceptible to minocycline and co-trimoxazole. Pulsed-field gel electrophoresis analysis revealed 4 shared strains among 17 CF patients in one pediatric clinic, but epidemiological investigations did not support patient-to-patient transmission except between one sibling pair.

Utility of real-time amplification of selected 16S rRNA gene sequences as a tool for detection and identification of microbial signatures directly from clinical samples.

The potential of incorporating a real-time PCR for amplification and detection of 16S rRNA gene signatures directly from clinical samples was assessed as a tool for diagnostics. Universal PCR primers spanning short variable regions (~500 bp) were optimized for real-time PCR and tested in comparison with a longer fragment (~1400 bp) generated from block-based amplification. Real-time PCR had improved sensitivity of detection (8% increase), decreased amplification time and simplified downstream processing. The real-time PCR primers also offered an improvement in detection of bacteria from samples that demonstrated inhibition with the block-based primers and in the resolution of mixed-sequence traces. In addition to testing primer sensitivity, the effect of amplifying and sequencing two different variable regions of the 16S rRNA gene on organism identification was compared. By amplifying and sequencing a shorter variable region, the number of species that were identified to the species level was reduced by 18%.

Flagellin gene sequence evolution in Salmonella.

Salmonella exhibits 70 serologically distinct flagellins, used internationally to diagnose and track infections. The terminal sequences of flagellin protein subunits are conserved in a range of bacteria and are here used as evolutionary markers to reveal how new serotypes arise. Terminal sequences of flagellins that exhibit factors g or m (G-group) were distinct from other Salmonella antigens (Non-G-group) and cluster more closely with Escherichia coli. It is postulated that G-group flagellins were inherited from a common ancestor of E. coli and Salmonella and that these antigens were among the original set in Salmonella. Sequence differences at the 5' termini may prevent recombination between co-infecting strains. Evidence of increased variation of flagellin in rare biphasic G-group serotypes suggests that the presence of a second flagellin locus allows mutation of the G-group flagellin. FljB probably arose from a single duplication of a Non-G gene, since which synonymous mutations resulted in the fljB-specific sequence at the 5' termini.

Towards the development of a DNA-sequence based approach to serotyping of Salmonella enterica.

BACKGROUND: The fliC and fljB genes in Salmonella code for the phase 1 (H1) and phase 2 (H2) flagellin respectively, the rfb cluster encodes the majority of enzymes for polysaccharide (O) antigen biosynthesis, together they determine the antigenic profile by which Salmonella are identified. Sequencing and characterisation of fliC was performed in the development of a molecular serotyping technique. RESULTS: FliC sequencing of 106 strains revealed two groups; the g-complex included those exhibiting "g" or "m,t" antigenic factors, and the non-g strains which formed a second more diverse group. Variation in fliC was characterised and sero-specific motifs identified. Furthermore, it was possible to identify differences in certain H antigens that are not detected by traditional serotyping. A rapid short sequencing assay was developed to target serotype-specific sequence motifs in fliC. The assay was evaluated for identification of H1 antigens with a panel of 55 strains. CONCLUSION: FliC sequences were obtained for more than 100 strains comprising 29 different H1 alleles. Unique pyrosequencing profiles corresponding to the H1 component of the serotype were generated reproducibly for the 23 alleles represented in the evaluation panel. Short read sequence assays can now be used to identify fliC alleles in approximately 97% of the 50 medically most important Salmonella in England and Wales. Capability for high throughput testing and automation give these assays considerable advantages over traditional methods.

Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing.

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to 276 Campylobacter jejuni strains and 87 Campylobacter coli strains isolated from humans, pigs, cattle, poultry, and retail meats to investigate whether certain FAFLP genotypes of C. jejuni and C. coli are associated with a particular host and to determine the degree of association between FAFLP-defined genotypes and heat-stable serotypes and/or phage types. Within C. coli, the poultry strains clustered separately from those of porcine origin. In contrast, no evidence of host specificity was detected among C. jejuni strains. While C. coli strains show host specificity by FAFLP genotyping, C. jejuni strains that are genotypically similar appear to colonize a range of hosts, rather than being host adapted. Some serotypes and/or phage types (C. jejuni serotype HS18, phage type PT6, and serophage type HS19/PT2 and C. coli HS66, PT2, and HS56/PT2) were the most homogeneous by FAFLP genotyping, while others were more heterogeneous (C. jejuni HS5 and PT39, and C. coli HS24 and PT44) and therefore poor indicators of genetic relatedness between strains. The lack of host specificity in C. jejuni suggests that tracing the source of infection during epidemiological investigations will continue to be difficult. The lack of congruence between some serotypes and/or phage types and FAFLP genotype underlines the need for phenotypic testing to be supplemented by genotyping. This study also demonstrates how, in general, FAFLP generates "anonymous" genetic markers for strain characterization and epidemiological investigation of Campylobacter in the food chain.