Eleanor McNamara

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.

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis. Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3-26.6% for tetracycline and 33.3-36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.

Antibiotic resistance of retail food and human Campylobacter isolates on the island of Ireland from 2001-2002.

The antimicrobial resistance profiles of Campylobacter isolates recovered from a range of retail food samples (n=374) and humans (n=314) to eight antimicrobial compounds were investigated. High levels of resistance in food C. jejuni isolates were observed for ceftiofur (58%), ampicillin (25%) and nalidixic acid (17%) with lower levels observed for streptomycin (7.9%) and chloramphenicol (8.3%). A total of 80% of human C. jejuni isolates were resistant to ceftiofur, while 17% showed resistance to ampicillin and nalidixic acid, 8.6% to streptomycin and 4.1% to chloramphenicol. Resistance to clinically relevant antimicrobials such as erythromycin, ciprofloxacin and tetracycline was 6.7, 12, and 15% respectively for all food isolates and was similar to corresponding resistance prevalences observed for human isolates, where 6.4, 12 and 13% respectively were found to be resistant. Comparisons of C. jejuni isolates in each location showed a high degree of similarity although some regional variations did exist. Comparison of total C. jejuni and C. coli populations showed minor differences, with C. jejuni isolates more resistant to ampicillin and ceftiofur. Multidrug resistance patterns showed some profiles common to human and clinical isolates.

Occurrence and characteristics of fastidious Campylobacteraceae species in porcine samples.

This study investigated the prevalence and characteristics of Campylobacteraceae including a range of fastidious species in porcine samples. Over a thirteen month period caecal contents (n=402) and pork carcass swabs (n=401) were collected from three pork abattoirs and pork products (n=399) were purchased at point of sale in the Republic of Ireland. Campylobacteraceae isolates were recovered by enrichment, membrane filtration and incubation in antibiotic free media under a modified atmosphere (3% O2, 5% H2, 10% CO2 and 82% N2). Campylobacteraceae isolates were identified as either genus Campylobacter or Arcobacter and then selected species were identified by Polymerase Chain Reaction (PCR). Campylobacteraceae were isolated from 103 (26%) caecal samples, 42 (10%) carcass swabs, and 59 (15%) pork products. Campylobacter coli was the most commonly isolated species found in (37%) all sample types but many fastidious species were also isolated including Campylobacter concisus (10%), Arcobacter butzleri (8%), Campylobacter helveticus (8%), Campylobacter mucosalis (6%), Arcobacter cryaerophilus (3%), Campylobacter fetus subsp. fetus (1%), Campylobacter jejuni subsp. jejuni (1%), Campylobacter lari (0.5%), Campylobacter curvus (0.5%) and Arcobacter skirrowii (0.5%). Among all isolates, 83% contained cadF and 98% flaA. In this study 35% of porcine C. coli were resistant to ciprofloxacin but none of the fastidious species demonstrated any resistance to this drug. The level of resistance to erythromycin was very high (up to 100%) in C. concisus and C. helveticus and this is a real concern as this is the current empiric drug of choice for treatment of severe gastroenteritic Campylobacter infections. The study shows that there is a much wider range of fastidious Campylobacteraceae present in porcine samples than previously assumed with C. concisus the second most common species isolated. The majority of fastidious Campylobacteraceae isolates obtained contained virulence genes and antibiotic resistance indicating potential public health significance.

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin.

Aims: To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin.

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 authors 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.

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.

Verotoxigenic Escherichia coli transmission in Ireland: a review of notified outbreaks, 2004–2012

Verotoxigenic Escherichia coli (VTEC) are significant for their low infectious dose, their potential clinical severity and the frequency with which they generate outbreaks. To describe the relative importance of different outbreak transmission routes for VTEC infection in Ireland, we reviewed outbreak notification data for the period 2004-2012, describing the burden and characteristics of foodborne, waterborne, animal contact and person-to-person outbreaks. Outbreaks where person-to-person spread was reported as the sole transmission route accounted for more than half of all outbreaks and outbreaks cases, most notably in childcare facilities. The next most significant transmission route was waterborne spread from untreated or poorly treated private water supplies. The focus for reducing incidence of VTEC should be on reducing waterborne and person-to-person transmission, by publicizing Health Service Executive materials developed for consumers on private well management, and for childcare facility managers and public health professionals on prevention of person-to-person spread.

Serogroup-specific Seasonality of Verotoxigenic Escherichia coli, Ireland.

To the Editor: Globally, an increasing number of serogroups of verotoxigenic Escherichia coli (VTEC) have been reportedly associated with human illness. The best known is serogroup O157; the World Health Organization also recognizes VTEC O103, O111, O145, and O26 as having the potential to cause severe disease (1). The increasing number of non-O157 VTEC infections is cause for concern. In general, human infections with VTEC are reportedly more common in late summer; the European Centre for Disease Control and Prevention reported that the number of cases across the European Union peaks each year during July–September (2). Similarly, the United States reported that the number of VTEC O157 cases peaks in late summer (3). Ireland is now one of the countries with the highest incidence of VTEC infection (2). In Ireland, statutory notification of VTEC infection became mandatory in 2004. In common with surveillance internationally, the focus was initially on VTEC O157; since then, testing and surveillance for non-O157 VTEC have improved substantially as a result of increased awareness and availability of diagnostic methods for non-O157 detection. Non-O157 VTEC were first reported in Ireland in 1999 (4), and surveillance data indicated that only 14% of VTEC notifications in 2004 compared with 75% in 2014 were caused by non-O157 VTEC. In the notification dataset for Ireland, the 2 primary VTEC serogroups (O26 and O157) over many years have seemed to differ in their seasonality; VTEC O26 notifications generally peaked ≈2 months earlier than VTEC O157 notifications (Figure, panel A). This earlier incidence peak for VTEC O26 has become progressively more consistent as the number of reported VTEC O26 notifications has risen. A study by Rivero et al. also suggested that non-O157 human infections may not exhibit the same seasonal variation observed for VTEC O157 (5). Figure Verotoxigenic Escherichia coli (VTEC) O157 and VTEC O26, Ireland, 2004–2014. A) Seasonal distribution of notifications. B) Predicted seasonal distribution. Data source: Computerised Infectious Disease Reporting System (https://www.hpsc.ie/NotifiableDiseases ... In this study, we compared the seasonality of the 2 strains by using national notification data for 2004–2014 (n = 2,569 notifications for O157 and O26). We estimated the timing of the seasonal peaks (phase of seasonality) for each of the serogroups, and the difference between the 2 phases, by using times series quasi-Poisson regression, fitting terms for temporal trend, and a sine wave with a period of 12 months for seasonality and for interaction by serogroup. We compared the phase shifts of the 2 serogroups by using the Wald test. To rule out the possibility that the observed distributions were influenced by the occurrence of a limited number of outbreaks, we reanalyzed the data for sporadic cases alone and, because risk factors for VTEC infection have been shown to vary by age (6), separately for patients <5 years of age and for older child and adult patients. The number of predicted cases peaked in July for VTEC O26 and in September for VTEC O157; the 2-month difference in phase (seasonality) by serogroup was significant (p 5 years of age (predicted difference in phase 1 month, p<0.0001). A significant increasing annual trend was also observed, in particular for VTEC O26. However, this increase is probably, at least in part, artifactual because of increased availability and more widespread use of clinical diagnostic tests for non-O157 VTEC in later years. One possible explanation for the difference in seasonality is that the primary animal reservoirs for the 2 serogroups could differ. Cattle and sheep have been identified as carriers of O157 and O26 strains in Ireland (7,8). In Germany, cattle density has been shown to be significantly associated with human VTEC O157 incidence but only marginally associated with O26 incidence (9); the same study showed no association between cattle density and VTEC O91 infection, indicating that not all serogroups necessarily share the same reservoirs. Alternatively, animals of the same species may be preferentially colonized with different serogroups at different times of the year or at different developmental ages. Other possible explanations could be variation in survival characteristics between the 2 strains, which results in a different seasonal distribution in the environment, or specific human behavior (e.g., seasonal food) resulting in more frequent exposure to sources of VTEC O157 and VTEC O26 at different times of the year. The consistent differences in seasonality identified here between the 2 most common VTEC serogroups suggest the existence of noteworthy underlying differences in disease etiology between the strains. Further exploration is recommended.

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.