Molecular diagnostics: the changing culture of medical microbiology

Diagnostic molecular biology is arguably the fastest growing area in current laboratory-based medicine. Growth of the so called ‘omics’ technologies has, over the last decade, led to a gradual migration away from the ‘one test, one pathogen’ paradigm, toward multiplex approaches to infectious disease diagnosis, which have led to significant improvements in clinical diagnostics and ultimately improved patient care

Changing Diagnostic Methods and Increased Detection of Verotoxigenic Escherichia coli, Ireland

The recent paradigm shift in infectious disease diagnosis from culture-based to molecular-based approaches is exemplified in the findings of a national study assessing the detection of verotoxigenic Escherichia coli infections in Ireland. The methodologic changes have been accompanied by a dramatic increase in detections of non-O157 verotoxigenic E. coli serotypes

Genomic Investigation into Strain Heterogeneity and Pathogenic Potential of the Emerging Gastrointestinal Pathogen Campylobacter ureolyticus

The recent detection and isolation of C. ureolyticus from patients with diarrhoeal illness and inflammatory bowel diseases warrants further investigation into its role as an emerging pathogen of the human gastrointestinal tract. Regarding the pathogenic mechanisms employed by this species we provide the first whole genome analysis of two C. ureolyticus isolates including the type strain. Comparative analysis, subtractive hybridisation and gene ontology searches against other Campylobacter species identifies the high degree of heterogenicity between C. ureolyticus isolates, in addition to the identification of 106 putative virulence associated factors, 52 of which are predicted to be secreted. Such factors encompass each of the known virulence tactics of pathogenic Campylobacter spp. including adhesion and colonisation (CadF, PEB1, IcmF and FlpA), invasion (ciaB and 16 virB-virD4 genes) and toxin production (S-layer RTX and ZOT). Herein, we provide the first virulence catalogue for C. ureolyticus, the components of which theoretically provide this emerging species with sufficient arsenal to establish pathology

Investigation of molecular mechanisms underlying tetracycline resistance in thermophilic Campylobacter spp. suggests that previous reports of tet(A)-mediated resistance in these bacteria are premature

peer-reviewedThe true prevalence of tet(A), which codes for a tetracycline efflux pump, in thermophilic Camplyobacter spp. requires clarification after reports emerged in Iran (2014) and Kenya (2016) of the novel detection of tet(A) in Campylobacter. During our investigation of antibiotic resistance mechanisms in a sample of Irish thermophilic Campylobacter broiler isolates, it was determined that 100% of tetracycline-resistant isolates (n = 119) harboured tet(O). Accessory tetracycline-resistance mechanisms were considered as tetracycline minimum inhibitory concentrations ranged from 4 to ≥ 64 mg/L. Primers previously reported for the detection of tet(A) in Campylobacter failed to produce an amplicon using a positive control strain (Escherichia coli K12 SK1592 containing the pBR322 plasmid) and a selection of Campylobacter isolates. Accordingly, we designed new tet(A)-targeting primers on SnapGene2.3.2 that successfully generated a 407 bp product from the positive control strain only. Further in silico analysis using BLASTn and SnapGene2.3.2 revealed that previously reported Campylobacter tet(A) sequences deposited on GenBank shared 100% homology with Campylobacter tet(O). We postulate that this gave rise to the erroneous report of a high tet(A) prevalence among a pool of Kenyan broiler Campylobacter isolates that were tested using primers designed based on these apparent tet(A) sequences. In conclusion, further work would be required to determine whether the homology between tet(A) potentially present in Campylobacter and known tet(A) genes would be sufficient to allow amplification using the primers designed in our study. Finally, the existence of tet(A) in thermophilic Campylobacter spp. remains to be demonstrated

Campylobacter ureolyticus: an emerging gastrointestinal pathogen?

A total of 7194 faecal samples collected over a 1-year period from patients presenting with diarrhoea were screened for Campylobacter spp. using EntericBios, a multiplex-PCR system. Of 349 Campylobacter-positive samples, 23.8% were shown to be Campylobacter ureolyticus, using a combination of 16S rRNA gene analysis and highly specific primers targeting the HSP60 gene of this organism. This is, to the best of our knowledge, the first report of C. ureolyticus in the faeces of patients presenting with gastroenteritis and may suggest a role for this organism as an emerging enteric pathogen

Draft Genome Sequence of Campylobacter fetus subsp. fetus CITCf01, Isolated from a Patient with Subacute Bacterial Endocarditis

Campylobacter fetus is a Gram-negative, zoonotic pathogen and a member of the class Epsilonproteobacteria. We report the draft genome sequence of C. fetus subsp. fetus CITCf01, isolated from a patient with subacute bacterial endocarditis. CITCf01 grew under aerobic, microaerobic, and anaerobic conditions, and at 42°C, an unusual combination of growth conditions

Emerging dynamics of human campylobacteriosis in Southern Ireland

Infections with Campylobacter spp. pose a significant health burden worldwide. The significance of Campylobacter jejuni/Campylobacter coli infection is well appreciated but the contribution of non-C. jejuni/C. coli spp. to human gastroenteritis is largely unknown. In this study, we employed a two-tiered molecular study on 7194 patient faecal samples received by the Microbiology Department in Cork University Hospital during 2009. The first step, using EntericBio® (Serosep), a multiplex PCR system, detected Campylobacter to the genus level. The second step, utilizing Campylobacter species-specific PCR identified to the species level. A total of 340 samples were confirmed as Campylobacter genus positive, 329 of which were identified to species level with 33 samples containing mixed Campylobacter infections. Campylobacter jejuni, present in 72.4% of samples, was the most common species detected, however, 27.4% of patient samples contained non-C. jejuni/C. coli spp.; Campylobacter fetus (2.4%), Campylobacter upsaliensis (1.2%), Campylobacter hyointestinalis (1.5%), Campylobacter lari (0.6%) and an emerging species, Campylobacter ureolyticus (24.4%). We report a prominent seasonal distribution for campylobacteriosis (Spring with C. ureolyticus (March) preceeding slightly C. jejuni/C. coli (April/May)

Integronlike Structures in Campylobacter spp. of Human and Animal Origin

Resistance to antimicrobial agents used to treat severe Campylobacter spp. gastroenteritis is increasing worldwide. We assessed the antimicrobial resistance patterns of Campylobacter spp. isolates of human and animal origin. More than half (n = 32) were resistant to sulphonamide, a feature known to be associated with the presence of integrons. Analysis of these integrons will further our understanding of Campylobacter spp. epidemiology

Antimicrobial Resistance Determinants Circulating among Thermophilic Campylobacter Isolates Recovered from Broilers in Ireland Over a One-Year Period

peer-reviewedCampylobacteriosis is the leading cause of human bacterial gastroenteritis, very often associated with poultry consumption. Thermophilic Campylobacter (Campylobacter jejuni and Campylobacter coli) isolates (n = 158) recovered from broiler neck skin and caecal contents in Ireland over a one-year period, resistant to at least one of three clinically relevant antimicrobial classes, were screened for resistance determinants. All ciprofloxacin-resistant isolates (n = 99) harboured the C257T nucleotide mutation (conferring the Thr-86-Ile substitution) in conjunction with other synonymous and nonsynonymous mutations, which may have epidemiological value. The A2075G nucleotide mutation and amino acid substitutions in L4 and L22 were detected in all erythromycin-resistant isolates (n = 5). The tetO gene was detected in 100% (n = 119) of tetracycline-resistant isolates and three of which were found to harbour the mosaic tetracycline resistance gene tetO/32/O. Two streptomycin-resistant C. jejuni isolates (isolated from the same flock) harboured ant(6)-Ib, located in a multidrug resistance genomic island, containing aminoglycoside, streptothricin (satA) and tetracycline resistance genes (truncated tetO and mosaic tetO/32/O). The ant(6)-Ie gene was identified in two streptomycin-resistant C. coli isolates. This study highlights the widespread acquisition of antimicrobial resistance determinants among chicken-associated Campylobacter isolates, through horizontal gene transfer or clonal expansion of resistant lineages. The stability of such resistance determinants is compounded by the fluidity of mobile genetic element

Testing barrier materials in the development of a biosecurity pen to protect broilers against Campylobacter

peer-reviewedPrevious studies demonstrated that commercial broiler flocks could be protected from Campylobacter colonisation using a bird pen, termed the “biosecurity cube”, constructed from four polycarbonate sheets (1m high x 2.5m long x 6 mm thick) supported at the corners by 4 × 1m high wooden columns. However, this design had issues with airflow and potential for upscaling. A biosecurity cube composed of four galvanised steel mesh panels (3.44m long x 1.25m high) was therefore developed onto which different barrier materials, preventing contact between the test birds and the main flock, were attached. The objective of this study was to test a range of barrier materials including cardboard, wire mesh, polyurethane film and later (at the suggestion of broiler industry personnel) flyscreen mesh. Initial studies suggested that while the cardboard and wire mesh were ineffective, the polyurethane film protected the birds. Further validation (over 2 separate trials, 7 cubes for each barrier material) demonstrated that polyurethane and flyscreen mesh were effective. It was concluded that a biosecurity pen infrastructure based on galvanised steel mesh panels surrounded by polyurethane film or flyscreen mesh was effective at protecting the birds from Campylobacter but upscaling studies will be undertaken before full implementation