Genotypic and phenotypic antimicrobial resistance of Irish Clostridioides difficile isolates, 2022.

OBJECTIVES: Infection with Clostridioides difficile (C. difficile) usually occurs after antibiotic treatment for other infections and can cause gastro-intestinal disorders of variable severity. C. difficile can be resistant to a wide spectrum of antimicrobials. Detection of antimicrobial resistance (AMR) is important to direct optimal treatment and surveillance of AMR patterns in the overall population. Correlation between genotypic markers and phenotypic AMR is not yet well defined. The aim for this study is to assess whether and to what extent genotypic determinants of AMR correlate with phenotypic resistance. METHODS: 99 C. difficile isolates were phenotypically characterized for resistance to eight antibiotics using Sensititre plates or E-tests. Their genomes were screened for genetic markers of resistance. Accuracy, sensitivity, specificity, positive and negative predictive values were calculated. RESULTS: We found high rates of resistance (>50%) to cefoxitin and clindamycin, intermediate rates of resistance (10% - 50%) to moxifloxacin and tetracycline and low rates of resistance (<10%) to imipenem, metronidazole, vancomycin, and rifampicin. For moxifloxacin, tetracycline, and clindamycin, we found a good correlation between genotypic and phenotypic AMR, with an overall accuracy of 96% (95% CI 90%-100%), 78% (95% CI 68%-86%) and 86% (95% CI 77%-92%) respectively. For the other five antibiotics, accurate estimates on the correlation could not be made. CONCLUSION: Our results suggest that for moxifloxacin, tetracycline and clindamycin, phenotypic resistance in C. difficile can be predicted by genetic indicators and used for public health purposes. However, for the other five antibiotics, the model is not accurate and further development is necessary.

Shiga toxin-producing Escherichia coli clonal complex 32, including serotype O145:H28, in the UK and Ireland.

Introduction. Shiga toxin-producing Escherichia coli (STEC) O157:H7 has been the most clinically significant STEC serotype in the UK for over four decades. Over the last 10 years we have observed a decrease in STEC O157:H7 and an increase in non-O157 STEC serotypes, such as O145:H28.Gap Statement. Little is known about the microbiology and epidemiology of STEC belonging to CC32 (including O145:H28) in the UK. The aim of this study was to integrate genomic data with patient information to gain a better understanding of the virulence, disease severity, epidemic risk assessment and population structure of this clinically significant clonal complex.Methodology. Isolates of E. coli belonging to CC32 (n=309) in the archives of public health agencies in the UK and Ireland were whole-genome-sequenced, virulence-profiled and integrated with enhanced surveillance questionnaire (ESQ) data, including exposures and disease severity.Results. Overall, diagnoses of STEC belonging to CC32 (290/309, 94 %) in the UK have increased every year since 2014. Most cases were female (61 %), and the highest proportion of cases belonged to the 0-4 age group (53/211,25 %). The frequency of symptoms of diarrhoea (92 %), abdominal pain (84 %), blood in stool (71 %) and nausea (51 %) was similar to that reported in cases of STEC O157:H7, although cases of STEC CC32 were more frequently admitted to hospital (STEC CC32 48 % vs O157:H7  34 %) and/or developed haemolytic uraemic syndrome (HUS) (STEC CC32 9 % vs O157:H7 4 %).The majority of STEC isolates (268/290, 92 %) had the stx2a/eae virulence gene combination, most commonly associated with progression to STEC HUS. There was evidence of person-to-person transmission and small, temporally related, geographically dispersed outbreaks, characteristic of foodborne outbreaks linked to nationally distributed products.Conclusion. We recommend more widespread use of polymerase chain reaction (PCR) for the detection of all STEC serogroups, the development of consistent strategies for the follow-up testing of PCR-positive faecal specimens, the implementation of more comprehensive and standardized collection of epidemiological data, and routine sharing of sequencing data between public health agencies worldwide.

Comparison and correlation of commercial SARS-CoV-2 real-time-PCR assays, Ireland, June 2020.

We report the performance of a variety of commercially available SARS-CoV-2 PCR kits, used in several different sites across Ireland to determine if Ct values across platforms are comparable. We also investigate whether a Ct value, a surrogate for calculated viral loads in the absence of viral culture of > 34 can be used to exclude SARS-CoV-2 infection and its complications. We found a variation in Ct values from different assays for the same calculated viral load; this should be taken into consideration for result interpretation.

Establishment of sentinel surveillance of human clinical campylobacteriosis in Ireland.

The aim of this work was the establishment of a national laboratory sentinel surveillance service for human clinical Campylobacter in Ireland. This included detailed genomic molecular epidemiology of Campylobacter for 2019. For February-December 2019, 24 clinical microbiology laboratories in Ireland submitted all PCR/culture-positive clinical Campylobacter spp. specimens to Public Health Laboratory (PHL) Dublin one week out of every four. Antimicrobial susceptibility testing (AST) according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria was carried out for Campylobacter spp. isolates for ciprofloxacin, tetracycline and erythromycin. Batch whole genome sequencing (WGS) was carried out on cultures and analysis was performed to determine species, genotype, identify antimicrobial resistance (AMR) and virulence determinants and identify clusters. A total of 75 isolates and 366 PCR-positive stools were received, and 277 isolates recovered (55.7% recovery from stools). Of 257 isolates characterized by WGS, 86.4% (n = 222) were Campylobacter jejuni, 11.7% (n = 30) Campylobacter coli and 1.9% (n = 5) Campylobacter lari. There were 20 clonal complexes with ST-21 clonal complex most prevalent at 26.8% (n = 69). 50.5% (n = 140) of isolates were susceptible to all three antimicrobials tested. 39.3% (n = 109) isolates were ciprofloxacin resistant, 26.3% (n = 73) tetracycline resistant and two isolates erythromycin resistant. Congruence between phenotypic and genotypic AST was observed. There was 95.9% and 95.6% sensitivity and specificity for WGS to predict ciprofloxacin sensitivity and 98.6% and 99.5% sensitivity and specificity for WGS to predict tetracycline sensitivity. Virulence factors flaA, racR, ciaB and cdtB were detected in all isolates. WGS identified 31 potential clusters for public health alert. This sentinel surveillance of human campylobacteriosis in Ireland establishes the basis for a national reference service. Linking with other partners in a 'One Health' framework will help us better understand sources of infection to reduce disease burden and the threat of AMR.

Molecular epidemiology of an extended multiple-species OXA-48 CPE outbreak in a hospital ward in Ireland, 2018-2019.

Objectives: Molecular epidemiological description of an OXA-48 CPE outbreak affecting a tertiary-care hospital ward in Ireland over an extended period (2018-2019). Methods: Microbiological testing and whole-genome sequencing (WGS) were performed on all 56 positive OXA-48 outbreak case isolates. Results: In total, 7 different species were identified: Enterobacter hormaechei (n = 35, 62.5%), Escherichia coli (n = 12, 21.4%), Klebsiella pneumoniae (n = 5, 8.9%), Klebsiella oxytoca (n = 1, 1.8%), Klebsiella michiganensis (n = 1, 1.8%), Citrobacter freundii (n = 1, 1.8%), and Serratia marcesens (n = 1, 1.8%). E. hormaechei ST78 was the most common genotype (n = 14, 25%). Two major pOXA-48 plasmid types were identified throughout the outbreak, 'types' 1 and 2, and 5 major E. hormaechei clonal groupings were identified: ST78, ST108, ST1126, ST135, and ST66. Within each of the ST108, ST1126, ST135 and ST66 groups, the pOXA-48 harbored within each isolate were the same. Within ST78, 9 isolates contained the pOXA48 'type 2' plasmid and 5 contained the 'type 1' plasmid. Environmental specimens were taken from different outbreak ward locations: handwash basins, sink and shower drains, and taps. Of 394 environmental specimens, OXA-48 CPE was isolated from 26 (6.6%). Conclusions: This prolonged outbreak of OXA-48 CPE was confined to one ward, but it exemplifies the complexity and difficulty in the control of these organisms. With multiple species and genotypes involved, they may be better described as 'plasmid outbreaks.' WGS provided insights into this diversity and potential transmission among cases, though its usefulness would be enhanced by analysis as close as possible to real time so that interventions can be implemented as soon as data are available.

Neuraminidase characterisation reveals very low levels of antiviral resistance and the presence of mutations associated with reduced antibody effectiveness in the Irish influenza 2018/2019 season.

Neuraminidase inhibitor (NAI) resistance levels globally are currently low. However, as antivirals are increasingly being used, and even in the absence of selective pressure, resistance may increase or emerge. The neuraminidase (NA) genes from influenza viruses from the Irish 2018/2019 season were sequenced: 1/144 (0.7 %) A(H1N1)pdm09 sequences harboured a substitution associated with highly-reduced susceptibility to NAIs. The very low NAI resistance we describe supports current Irish NAI use recommendations. However, continued monitoring is essential. NA characterisation also identified substitutions associated with reduced antibody effectiveness, thereby highlighting the potential of NA sequence surveillance as an additional tool for investigating influenza vaccine effectiveness (VE).

Genomic Analysis of Mycobacterium abscessus Complex Isolates Collected in Ireland between 2006 and 2017.

Members of the Mycobacterium abscessus complex (MABC) are multidrug-resistant nontuberculous mycobacteria and cause opportunistic pulmonary infections in individuals with cystic fibrosis (CF). In this study, genomic analysis of MABC isolates was performed to gain greater insights into the epidemiology of circulating strains in Ireland. Whole-genome sequencing (WGS) was performed on 70 MABC isolates that had been referred to the Irish Mycobacteria Reference Laboratory between 2006 and 2017 across nine Irish health care centers. The MABC isolates studied comprised 52 isolates from 27 CF patients and 18 isolates from 10 non-CF patients. WGS identified 57 (81.4%) as M. abscessus subsp. abscessus, 10 (14.3%) as M. abscessus subsp. massiliense, and 3 (4.3%) as M. abscessus subsp. bolletii Forty-nine (94%) isolates from 25 CF patients were identified as M. abscessus subsp. abscessus, whereas 3 (6%) isolates from 2 CF patients were identified as M. abscessus subsp. massiliense Among the isolates from non-CF patients, 44% (8/18) were identified as M. abscessus subsp. abscessus, 39% (7/18) were identified as M. abscessus subsp. massiliense, and 17% (3/18) were identified as M. abscessus subsp. bolletii WGS detected two clusters of closely related M. abscessus subsp. abscessus isolates that included isolates from different CF centers. There was a greater genomic diversity of MABC isolates among the isolates from non-CF patients than among the isolates from CF patients. Although WGS failed to show direct evidence of patient-to-patient transmission among CF patients, there was a predominance of two different strains of M. abscessus subsp. abscessus Furthermore, some MABC isolates were closely related to global strains, suggesting their international spread. Future prospective real-time epidemiological and clinical data along with contemporary MABC sequence analysis may elucidate the sources and routes of transmission among patients infected with MABC.

Antimicrobial Susceptibility of Clinical and Environmental Mycobacterium chimaera Isolates.

Mycobacterium chimaera is a slow-growing nontuberculous Mycobacterium species belonging to the Mycobacterium avium complex (MAC). It has been identified globally as the cause of a large outbreak of cardiovascular infections following open heart surgery, but it can also cause respiratory infections in individuals with underlying structural pulmonary disease. Invasive M. chimaera infections are associated with poor clinical responses, and the optimal antibiotic treatment regimen for these infections is not known. In this study, the drug susceptibility profiles of clinical and environmental M. chimaera isolates for antimicrobial agents that are commonly considered for treatment of MAC infections were determined. All M. chimaera isolates were susceptible to clarithromycin, with a median MIC of 2 µg/ml, while 98% (85/87 isolates) were susceptible to amikacin. Twenty-five percent of isolates (22/87 isolates) had intermediate susceptibility and 52% (46/87 isolates) were resistant to moxifloxacin. Similarly, 39% of isolates (34/87 isolates) had intermediate susceptibility and 39% (34/87 isolates) were resistant to linezolid. MIC breakpoints derived from the literature were used to determine resistance to rifampin (16/87 isolates [18%]), ethambutol (10/87 isolates [11%]), rifabutin (2/87 isolates [2%]), and streptomycin (1/87 isolates [1%]). In conclusion, our results showed that clarithromycin, amikacin, rifabutin, and streptomycin had the best activity against M. chimaera isolates, while susceptibility rates were lower for rifampin and ethambutol. In contrast, there was a high prevalence of isolates that were not susceptible to moxifloxacin or linezolid. While factors in addition to antibiotic susceptibility may determine the outcomes of treatment of M. chimaera infections, our results should inform the selection of antimicrobials as part of the overall therapeutic strategy.

Molecular characterization of Campylobacter causing human clinical infection using whole-genome sequencing: Virulence, antimicrobial resistance and phylogeny in Ireland.

OBJECTIVES: We characterized clinical isolates of Campylobacter using whole-genome sequencing (WGS) for detection of virulence genes, antimicrobial resistance markers and phylogenetic analysis in order to increase the knowledge on the molecular epidemiology of Campylobacter in Ireland, where there are significant gaps due to the widespread in the use of culture independent methods for the diagnosis of campylobacteriosis. METHODS: WGS was applied to 122 Campylobacter human isolates collected over a 10-years period, from diarrhoeal stool samples submitted for routine enteric screening. RESULTS: Genes associated with cytotoxin production such as cdtA, cdtB and cdtC were found in 88%, 89% and 89% isolates, respectively; adherence, colonization and invasion genes such as cadF, dnaJ, racR, iam, virB11 and ciaB were found in 99%, 99%, 98%, 99%, 1% and 80% isolates, respectively. Genetic markers associated with resistance to quinolones (C257T in gyrA), beta-lactams (blaoxa-61) and tetracycline (tet(O)) were present in 43%, 71% and 25% isolates, respectively. The cmeABC operon was present in 94% of isolates. No macrolide or aminoglycoside resistance markers were detected. Phylogenetic analysis showed that 112 isolates were assigned to 29 sequence types grouped into 17 clonal complexes. Four clusters previously unidentified were detected. These results shown the similarity of Irish data compared to what has been described globally. CONCLUSIONS: WGS has shown a high discriminatory power for cluster detection, demonstrating that its integration in routine laboratory surveillance could improve the detection and management of outbreaks. In addition we were able to demonstrate that virulence genes in clinical Campylobacter infections in Ireland were similar to those known previously. High prevalence of quinolone resistance markers has been found, which has implications for antimicrobial stewardship.

Evolutionary Context of Non-Sorbitol-Fermenting Shiga Toxin-Producing Escherichia coli O55:H7.

In July 2014, an outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 in England involved 31 patients, 13 (42%) of whom had hemolytic uremic syndrome. Isolates were sequenced, and the sequences were compared with publicly available sequences of E. coli O55:H7 and O157:H7. A core-genome phylogeny of the evolutionary history of the STEC O55:H7 outbreak strain revealed that the most parsimonious model was a progenitor enteropathogenic O55:H7 sorbitol-fermenting strain, lysogenized by a Shiga toxin (Stx) 2a-encoding phage, followed by loss of the ability to ferment sorbitol because of a non-sense mutation in srlA. The parallel, convergent evolutionary histories of STEC O157:H7 and STEC O55:H7 may indicate a common driver in the evolutionary process. Because emergence of STEC O157:H7 as a clinically significant pathogen was associated with acquisition of the Stx2a-encoding phage, the emergence of STEC O55:H7 harboring the stx2a gene is of public health concern.

Recurrent seasonal outbreak of an emerging serotype of Shiga toxin-producing Escherichia coli (STEC O55:H7 Stx2a) in the south west of England, July 2014 to September 2015.

The first documented British outbreak of Shiga toxin-producing Escherichia coli (STEC) O55:H7 began in the county of Dorset, England, in July 2014. Since then, there have been a total of 31 cases of which 13 presented with haemolytic uraemic syndrome (HUS). The outbreak strain had Shiga toxin (Stx) subtype 2a associated with an elevated risk of HUS. This strain had not previously been isolated from humans or animals in England. The only epidemiological link was living in or having close links to two areas in Dorset. Extensive investigations included testing of animals and household pets. Control measures included extended screening, iterative interviewing and exclusion of cases and high risk contacts. Whole genome sequencing (WGS) confirmed that all the cases were infected with similar strains. A specific source could not be identified. The combination of epidemiological investigation and WGS indicated, however, that this outbreak was possibly caused by recurrent introductions from a local endemic zoonotic source, that a highly similar endemic reservoir appears to exist in the Republic of Ireland but has not been identified elsewhere, and that a subset of cases was associated with human-to-human transmission in a nursery.

Evaluation of molecular and culture methods to determine the optimum testing strategy for verotoxigenic Escherichia coli in faecal specimens.

We evaluated 5 methods for the detection of verotoxogenic Escherichia coli (VTEC) from faecal specimens to determine the most sensitive and specific method(s) and to advise an optimum testing strategy. A total of 681 stool specimens were examined using up to 5 diagnostic molecular and phenotypic methods that are used routinely in the VTEC Reference laboratory, Dublin. A testing strategy incorporating a 2-step approach that included a single Real Time-PCR and 1 culture-based method yielded the highest sensitivity, specificity, positive predictive value, and negative predictive value of 98.21%, 100%, 100%, and 99.43%, respectively.

Characterization of farm, food, and clinical Shiga toxin-producing Escherichia coli (STEC) O113.

Thirty-nine Shiga toxin-producing Escherichia coli (STEC) O113 Irish farm, abattoir, and clinical isolates were analyzed in conjunction with eight Australian, New Zealand, and Norwegian strains for H (flagellar) antigens, virulence gene profile (eaeA, hlyA, tir, espA, espB katP, espP, etpD, saa, sab, toxB, iha, lpfA(O157/OI-141,) lpfA(O113,) and lpfA(O157/OI-154)), Shiga toxin gene variants (stx(1c), stx(1d), stx(2), stx(2c), stx(2dact), stx(2e), stx(2f,) and stx(2g)) and were genotyped using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All of the Irish strains were O113:H4, regardless of source, while all non-Irish isolates carried the H21 flagellar antigen. The stx(1) gene was present in 30 O113:H4 strains only, whereas the stx(2d) gene was common to all isolates regardless of source. In contrast, eaeA was absent, while hlyA was found in the Australian, New Zealand, Norwegian, and two of the Irish human clinical isolates. saa was present in the O113:H21 but not in the O113:H4 serotype. To the best of the author's knowledge, this is the first report of clinically significant STEC lacking both the eaeA and saa genes. PFGE analysis was inconclusive; however, MLST grouped the strains into three sequence types (ST): ST10, ST56, and ST223. Based on our findings, it was concluded that the stx(2d) gene is common in STEC O113, which are generally eaeA negative. Furthermore, STEC O113:H4 is a new, emerging bovine serotype of human clinical significance.

Multilocus sequence typing methods for the emerging Campylobacter Species C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus.

Multilocus sequence typing (MLST) systems have been reported previously for multiple food- and food animal-associated Campylobacter species (e.g., C. jejuni, C. coli, C. lari, and C. fetus) to both differentiate strains and identify clonal lineages. These MLST methods focused primarily on campylobacters of human clinical (e.g., C. jejuni) or veterinary (e.g., C. fetus) relevance. However, other, emerging, Campylobacter species have been isolated increasingly from environmental, food animal, or human clinical samples. We describe herein four MLST methods for five emerging Campylobacter species: C. hyointestinalis, C. lanienae, C. sputorum, C. concisus, and C. curvus. The concisus/curvus method uses the loci aspA, atpA, glnA, gltA, glyA, ilvD, and pgm, whereas the other methods use the seven loci defined for C. jejuni (i.e., aspA, atpA, glnA, gltA, glyA, pgm, and tkt). Multiple food animal and human clinical C. hyointestinalis (n = 48), C. lanienae (n = 34), and C. sputorum (n = 24) isolates were typed, along with 86 human clinical C. concisus and C. curvus isolates. A large number of sequence types were identified using all four MLST methods. Additionally, these methods speciated unequivocally isolates that had been typed ambiguously using other molecular-based speciation methods, such as 16S rDNA sequencing. Finally, the design of degenerate primer pairs for some methods permitted the typing of related species; for example, the C. hyointestinalis primer pairs could be used to type C. fetus strains. Therefore, these novel Campylobacter MLST methods will prove useful in differentiating strains of multiple, emerging Campylobacter species.

Genotypic characterisation and cluster analysis of Campylobacter jejuni isolates from domestic pets, human clinical cases and retail food.

The genetic similarity of Campylobacter jejuni isolates from pets, compared to human clinical cases and retail food isolates collected in Ireland over 2001-2006 was investigated by cluster analysis of pulsed-field gel electrophoresis (PFGE) fingerprinting profiles. Comparison of the PFGE profiles of 60 pet isolates and 109 human isolates revealed that seven (4.1%) profiles were grouped in clusters including at least one human and one pet C. jejuni isolate. In total six (1.6%) of 60 pet and 310 food profiles were in clusters with at least one food and one pet C. jejuni isolate. The detection of only a small number of genetically indistinguishable isolates by PFGE profile cluster analysis from pets and from humans with enteritis in this study suggests that pets are unlikely to be an important reservoir for human campylobacteriosis in Ireland. However, genetically indistinguishable isolates were detected and C. jejuni from pets may circulate and may contribute to clinical infections in humans. In addition, contaminated food fed to pets may be a potential source of Campylobacter infection in pets, which may subsequently pose a risk to humans.

Development and assessment of a rapid method to detect Escherichia coli O26, O111 and O157 in retail minced beef.

A molecular-based detection method was developed to detect Escherichia coli O26, O111 and O157 in minced (ground) beef samples. This method consists of an initial overnight enrichment in modified tryptone soya broth (mTSB) and novobiocin prior to DNA extraction and subsequent serogrouping using a triplex PCR. This method has a low limit of detection and results are available within 24 hours of receipt of samples. Once optimized, this rapid method was utilized to determine the prevalence of these E. coli serogroups in six hundred minced beef samples all of which were previously examined by immunomagnetic separation (IMS) and selective plating for E. coli O26 and O111. Using IMS, two E. coli O26 isolates were detected. No E. coli O111 were recovered. The multiplex PCR technique described here did not detect E. coli O111 nor O157 in any of the samples, however six minced beef samples were positive for E. coli O26 using our method, only two of these were previously detected by IMS and culture. Application of molecular methods are useful to support culture-based approaches thereby further contributing to risk reduction along the food chain.

Prevalence and characterization of Escherichia coli O26 and O111 in retail minced beef in Ireland.

Verocytotoxigenic Escherichia coli (VTEC) O157 are recognized as bacterial pathogens with significant public health impact. However, other serogroups, including O26, O111, O103, and O145, have the potential to cause the same spectrum of illness. In this study, 800 minced (ground) beef samples covering a large geographical region in Ireland were collected and tested for Escherichia coli (E. coli) O26 and E. coli O111 by conventional microbiological protocols. Two minced beef samples (0.25%) tested positive for E. coli O26, indicating fecal contamination. None of these isolates possessed verocytotoxin-encoding genes, (vt1/vt2 also known as stx1/stx2), the hemolysinencoding gene (hlyA), or the E. coli attachment-effacement (eae) gene, as determined by polymerase chain reaction (PCR). None of the beef samples analyzed contained E. coli O111. Although the E. coli O26 isolates were nonvirulent, the presence of these isolates in raw minced beef is an indication of fecal contamination and therefore potentially of public health significance.

Laboratory-based surveillance of human verocytotoxigenic Escherichia coli infection in the Republic of Ireland, 2002-2004.

The aim of this study was to examine the frequency and distribution of human verocytotoxigenic Escherichia coli (VTEC) O157 and non-O157 in the Republic of Ireland, and also to examine the presence of virulence genes in these isolates. This genetic information combined with phenotypic tests was used to produce a complete laboratory-based surveillance of human clinical VTEC infection in the Republic of Ireland between 2002 and 2004. Between January 2002 and December 2004 a total of 207 VTEC isolates were studied (one isolate per patient), 185 (89 %) of these were E. coli O157. The remaining 22 (11 %) were non-O157 E. coli, made up of 15 (7.2 %) E. coli O26, one (0.5 %) E. coli O103, one (0.5 %) E. coli O146, one (0.5 %) E. coli O145, two (1 %) E. coli O111 and two (1 %) ungroupable VTEC. These isolates originated from the eight health boards in the Republic of Ireland and represented over 90 % of the clinical cases of VTEC in the Republic of Ireland during this period. The results showed that VTEC O157 was the predominant serogroup and had a predominant toxin genotype of VT2 alone. Phage type 32 was the most common phage type of E. coli O157 identified. Non-O157 VTEC was a small proportion of all VTEC (10 % in 2002, 8 % in 2003, 15.5 % in 2004). In 2004 it was noted that there was an increase in the number and variety of non-O157 VTEC strains; however, this requires further monitoring in the future to see if this trend is sustained. It was also noted throughout the study period that the incidence of VTEC was higher in rural areas. Implementation of real-time PCR for the detection and subtyping of VTEC has aided outbreak investigations and is important for enhanced surveillance of VTEC in the Republic of Ireland.