Longitudinal carriage of antimicrobial resistant Enterobacterales in healthy individuals in Ireland - Assessing the impact of recreational water use on duration of carriage.

The increasing prevalence of extended-spectrum beta-lactamase (ESBL) producing Enterobacterales (ESBL-PE) and carbapenemase-producing Enterobacterales (CPE) is a major public health concern worldwide. Despite the associated risk of infection from gut colonisation with a resistant Enterobacterales, the incidence and duration of carriage in healthy individuals is poorly studied. This "persistence study" is the first in Ireland to assess the longitudinal carriage of ESBL-PE and CPE in healthy individuals. A cohort of 45 participants, 22 of whom were colonised with ESBL-PE, was recruited from a recently completed point prevalence study that investigated colonisation in recreational water users (WU) versus controls. Six bi-monthly faecal samples per participant were analysed for CPE and ESBL-PE over one year and the relationship between persistent colonisation and exposure to natural waters was investigated. For 11 of 45 participants (24.4 %) ESBL-E. coli (ESBL-EC) was detected in at least one sample. Genomic analysis revealed that six participants harboured the same ESBL-EC strains as identified in the preceding study. ESBL-EC persisted in the gut for a median duration of 10.3 months (range 4-23 months), consistent with previous research. Five participants (11.1 %) carried ESBL-EC for the entire study year. The carbapenemase gene blaIMI-2 was detected once. Colonisation was higher in water users during the non-bathing season (n = 10, November 2021-April 2022), than during the bathing season (n = 5, May 2022-September 2022) [relative risk 1.99 (95 % CI 0.34-11.71)]. However, overall WU were less likely to be colonised with ESBL-EC than controls (19 % vs 25 % respectively, RR 0.76, CI 0.24-2.34). Further research is warranted to better understand the factors influencing the persistence of gut colonisation with ESBL-EC and CPE and to what extent bathing water quality impacts colonisation for those regularly exposed.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Dissemination of carbapenemase-producing Enterobacterales in Ireland from 2012 to 2017: a retrospective genomic surveillance study.

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli.

First reported detection of the mobile colistin resistance genes, mcr-8 and mcr-9, in the Irish environment.

The emergence and dissemination of mobile colistin resistance (mcr) genes across the globe poses a significant threat to public health, as colistin remains one of the last line treatment options for multi-drug resistant infections. Environmental samples (157 water and 157 wastewater) were collected in Ireland between 2018 and 2020. Samples collected were assessed for the presence of antimicrobial resistant bacteria using Brilliance ESBL, Brilliance CRE, mSuperCARBA and McConkey agar containing a ciprofloxacin disc. All water and integrated constructed wetland influent and effluent samples were filtered and enriched in buffered peptone water prior to culture, while wastewater samples were cultured directly. Isolates collected were identified via MALDI-TOF, were tested for susceptibility to 16 antimicrobials, including colistin, and subsequently underwent whole genome sequencing. Overall, eight mcr positive Enterobacterales (one mcr-8 and seven mcr-9) were recovered from six samples (freshwater (n = 2), healthcare facility wastewater (n = 2), wastewater treatment plant influent (n = 1) and integrated constructed wetland influent (piggery farm waste) (n = 1)). While the mcr-8 positive K. pneumoniae displayed resistance to colistin, all seven mcr-9 harbouring Enterobacterales remained susceptible. All isolates demonstrated multi-drug resistance and through whole genome sequencing analysis, were found to harbour a wide variety of antimicrobial resistance genes i.e., 30 ± 4.1 (10-61), including the carbapenemases, blaOXA-48 (n = 2) and blaNDM-1 (n = 1), which were harboured by three of the isolates. The mcr genes were located on IncHI2, IncFIIK and IncI1-like plasmids. The findings of this study highlight potential sources and reservoirs of mcr genes in the environment and illustrate the need for further research to gain a better understanding of the role the environment plays in the persistence and dissemination of antimicrobial resistance.

A geographic cluster of healthcare-associated carbapenemase-producing hypervirulent Klebsiella pneumoniae sequence type 23.

Hypervirulent Klebsiella pneumoniae has typically been associated with invasive, community-associated infections. This study describes the molecular, epidemiological and clinical characteristics of a cluster of carbapenemase-producing hypervirulent K. pneumoniae in the South-East of Ireland. It highlights the increasing risk that hypervirulent K. pneumoniae poses to healthcare and residential care populations. A retrospective analysis of sequences on K. pneumoniae isolates in the K. pneumoniae database of the National Carbapenemase-Producing Enterobacterales Reference Laboratory Service was performed to identify cases of hypervirulent K. pneumoniae from one hospital network. Hypervirulence scores were assigned based on the presence of recognised hypervirulence genes. A retrospective review of patient records was carried out for all confirmed cases of hypervirulent K. pneumoniae identified and clinical, epidemiological and molecular characteristics described. Twenty-eight cases of hypervirulent OXA-48 producing K. pneumoniae were identified over a 2-year period. All isolates were sequence-type 23 with a hypervirulence score of 5. All isolates carried the blaOXA-48 carbapenemase gene. All cases had a record of current or recent hospitalisation or residence in a long-term residential care facility. This study describes extensive dissemination of hypervirulent K. pneumoniae within healthcare facilities and an ongoing outbreak in our region. It shows the convergence of hypervirulence and antibiotic resistance determinants. Healthcare facilities need to consider their infection prevention, control and surveillance strategies to monitor and prevent further dissemination among a vulnerable population. Diagnostic laboratories need to ensure they have the ability and capacity for testing. Readily deployed laboratory methods for detection of hypervirulence are required.

A Longitudinal Survey of Antibiotic-Resistant Enterobacterales in the Irish Environment, 2019-2020.

The natural environment represents a complex reservoir of antibiotic-resistant bacteria as a consequence of different wastewater discharges including anthropogenic and agricultural. Therefore, the aim of this study was to examine sewage and waters across Ireland for the presence of antibiotic-resistant Enterobacterales. Samples were collected from the West, East and South of Ireland. Two periods of sampling took place between July 2019 and November 2020, during which 118 water (30 L) and 36 sewage samples (200 mL) were collected. Waters were filtered using the CapE method, followed by enrichment and culturing. Sewage samples were directly cultured on selective agars. Isolates were identified by MALDI-TOF and antibiotic susceptibility testing was performed in accordance with EUCAST criteria. Selected isolates were examined for blaCTX-M, blaVIM, blaIMP, blaOXA-48, blaNDM, and blaKPC by real time PCR and whole genome sequencing (n = 146). A total of 419 Enterobacterales (348 water, 71 sewage) were isolated from all samples. Hospital sewage isolates displayed the highest percentage resistance to many beta-lactam and aminoglycoside antibiotics. Extended-spectrum beta-lactamase-producers were identified in 78% of water and 50% of sewage samples. One or more carbapenemase-producing Enterobacterales were identified at 23 individual sampling sites (18 water, 5 sewage). This included the detection of blaOXA-48 (n = 18), blaNDM (n = 14), blaKPC (n = 4) and blaOXA-484 (n = 1). All NDM-producing isolates harbored the ble-MBL bleomycin resistance gene. Commonly detected sequence types included Klebsiella ST323, ST17, and ST405 as well as E. coli ST131, ST38 and ST10. Core genome MLST comparisons detected identical E. coli isolates from wastewater treatment plant (WWTP) influent and nursing home sewage, and the surrounding waters. Similarly, one Klebsiella pneumoniae isolated from WWTP influent and the surrounding estuarine water were identical. These results highlight the need for regular monitoring of the aquatic environment for the presence of antibiotic-resistant organisms to adequately inform public health policies.

Case of recurrent Achromobacter xylosoxidans bacteraemia and PICC (peripherally-inserted central catheter) line infection in an immunocompromised patient.

BACKGROUND: This report describes recurrent A. xylosoxidans bloodstream and PICC (peripherally-inserted central catheter) line infection in an immunocompromised patient. PRESENTATION OF CASE: A 64-year-old female with acute promyelocytic leukaemia presented during a non-neutropenic febrile episode, and A. xylosoxidans was isolated from multiple PICC and peripheral blood cultures, and from the tip of the line on removal. The patient was treated with meropenem and a new PICC line was inserted after sterile blood cultures. Six weeks later, she represented with A. xylosoxidans from multiple cultures from the line. She was treated with piperacillin-tazobactam and the line was removed. There was no evidence of deep-seated infection. Further discussion revealed that the patient was using a sponge to clean, and a sleeve to cover her PICC-line while bathing. A. xylosoxidans was cultured from both the sponge and the swab. Whole Genome Sequencing performed on two blood culture isolated and both environmental isolates confirmed all four isolates were indistinguishable. The patient was advised not to use the sponge/sleeve in future and we have incorporated specific advice in this regard into our patient information. DISCUSSION: Achromobacter xylosoxidans is an aerobic, non-lactose fermenting gram-negative bacillus usually considered an opportunistic pathogen. It is associated with infection in immunocompromised patients, and is an emerging pathogen in catheter-related infections, sometimes associated with contaminated water. CONCLUSION: This case of recurrent A. xylosoxidans line infection highlights diagnostic and management challenges associated with catheter-related infections. Treatment is challenging because of intrinsic and acquired resistance mechanisms. Empiric treatment with anti-pseudomonal penicillins or carbapenems with line removal is typically required.

Genomic Characterization of Listeria monocytogenes Isolates Associated with Clinical Listeriosis and the Food Production Environment in Ireland.

Listeria monocytogenes is a major human foodborne pathogen that is prevalent in the natural environment and has a high case fatality rate. Whole genome sequencing (WGS) analysis has emerged as a valuable methodology for the classification of L. monocytogenes isolates and the identification of virulence islands that may influence infectivity. In this study, WGS was used to provide an insight into 25 L. monocytogenes isolates from cases of clinical infection in Ireland between 2013 and 2015. Clinical strains were either lineage I (14 isolates) or lineage II (11 isolates), with 12 clonal complexes (CC) represented, of which CC1 (6) and CC101 (4) were the most common. Single nucleotide polymorphism (SNP) analysis demonstrated that clinical isolates from mother-infant pairs (one isolate from the mother and one from the infant) were highly related (3 SNP differences in each) and also identified close similarities between isolates from otherwise distinct cases (1 SNP difference). Clinical strains were positive for common virulence-associated loci and 13 isolates harbour the LIPI-3 locus. Pulsed-field gel electrophoresis (PFGE) was used to compare strains to a database of 1300 Irish food and food processing environment isolates and determined that 64% of clinical pulsotypes were previously encountered in the food or food processing environment. Five of the matching food and food processing environment isolates were sequenced and results demonstrated a correlation between pulsotype and genotype. Overall, the work provides insights into the nature of L. monocytogenes strains currently causing clinical disease in Ireland and indicates that similar isolates can be found in the food or food processing environment.

Draft Genome Sequences of 25 Listeria monocytogenes Isolates Associated with Human Clinical Listeriosis in Ireland.

Listeria monocytogenes is a Gram-positive opportunistic pathogen that is the causative agent of listeriosis. Here, we report the draft genome sequences of 25 L. monocytogenes strains isolated from patients with clinical listeriosis in the Republic of Ireland between 2013 and 2015.

Comparison of Listeria monocytogenes isolates across the island of Ireland.

Building a comprehensive knowledge base of the association of Listeria monocytogenes isolates across national food chains, clinical cases, and environments can play a key role in helping control the incidence of listeriosis. Today, many food chains cross national borders and are often shared by neighboring countries. This study characterized L. monocytogenes isolated from food samples in Northern Ireland and investigated whether similarities in the population and associations of L. monocytogenes strains exist in the neighboring countries of Northern Ireland and the Republic of Ireland, which together constitute the island of Ireland. Listeria monocytogenes isolates were characterized using serotyping and pulsed-field gel electrophoresis subtyping. This data was then interrogated against existing data for the Republic of Ireland, to identify any shared trends in the ecology and contamination patterns of L. monocytogenes strains. The results of this study indicated that contaminated food products often shared L. monocytogenes strains with other products. A total of six different strain subtypes were identified among 18 contaminated products. Overall strain diversity in positive samples was low, with no sample yielding more than one L. monocytogenes strain, as determined by pulsed-field gel electrophoresis subtyping. When comparisons against an Irish strain database were performed, many related strain subtypes were also shared by a variety of sources in the Republic of Ireland. This study highlights the potential benefits that a whole-island surveillance approach may present to food safety and public health in both Northern Ireland and the Republic of Ireland.

PFGE analysis of Listeria monocytogenes isolates of clinical, animal, food and environmental origin from Ireland.

Listeria monocytogenes is an important foodborne human pathogen. Human infection is associated with high mortality rates. Epidemiological investigation and molecular subtyping can be useful in linking human illness with specific sources of infection. This retrospective study describes the use of PFGE to examine relationships of 222 isolates from human and non-human sources in Ireland. Human clinical isolates from other countries were also examined. Eight small clusters of human and non-human isolates (mostly serotype 4b) that were indistinguishable from one another were detected, suggesting potential sources for human infection. For non-human isolates, some PFGE types appeared to be exclusively associated with a single source, whereas other PFGE-types appeared to be more widely disseminated. Indistinguishable, or highly related clusters of isolates of Irish and non-Irish origin suggest that some PFGE patterns may be globally distributed.

Completeness and timeliness of Salmonella notifications in Ireland in 2008: a cross sectional study.

BACKGROUND: In Ireland, salmonellosis is the second most common cause of bacterial gastroenteritis. A new electronic system for reporting (Computerised Infectious Disease Reporting--CIDR) of Salmonella cases was established in 2004. It collates clinical (and/or laboratory) data on confirmed and probable Salmonella cases. The authors studied the completeness and the timeliness of Salmonella notifications in 2008. METHODS: This analysis was based upon laboratory confirmed cases of salmonella gastroenteritis. Using data contained in CIDR, we examined completeness for certain non-mandatory fields (country of infection, date of onset of illness, organism, outcome, patient type, and ethnicity). We matched the CIDR data with the dataset provided by the national Salmonella reference laboratory (NSRL) to which all Salmonella spp. isolates are referred for definitive typing. We calculated the main median time intervals in the flow of events of the notification process. RESULTS: In total, 416 laboratory confirmed Salmonella cases were captured by the national surveillance system and the NSRL and were included in the analysis. Completeness of non mandatory fields varied considerably. Organism was the most complete field (98.8%), ethnicity the least (11%). The median time interval between sample collection (first contact of the patient with the healthcare professional) to the first notification to the regional Department of Public Health (either a clinical or a laboratory notification) was 6 days (Interquartile 4-7 days). The median total identification time interval, time between sample collections to availability of serotyping and phage-typing results on the system was 25 days (Interquartile 19-32 days). Timeliness varied with respect to Salmonella species. Clinical notifications occurred more rapidly than laboratory notifications. CONCLUSIONS: Further feedback and education should be given to health care professionals to improve completeness of reporting of non-mandatory fields. The efficiency of reporting was similar to that published elsewhere. Delays in the reporting system at present mean that although the system is of value in facilitating comprehensive reporting it is unlikely it can be relied upon for rapid detection of outbreaks at an early stage. Direct person-to-person, communication between clinical and reference laboratories and public health practitioners remains a critical element of the surveillance system for rapid outbreak detection.

Interpreting streptomycin susceptibility test results for Salmonella enterica serovar Typhimurium.

Resistance or susceptibility of Salmonella enterica to streptomycin is widely used as an epidemiological marker. However, there is no clear consensus on the interpretation of streptomycin susceptibility test results. Comparison of results obtained with the Clinical and Laboratory Standards Institute (CLSI) disk diffusion method, the minimum inhibitory concentration (MIC) determined by Etest and streptomycin resistance genotype for 90 isolates of S. enterica serovar Typhimurium suggests that appropriate interpretive criteria for MIC results are susceptible at or=16 mg/L. For CLSI disk diffusion, we propose susceptible at a zone diameter>or=13 mm and resistant at

Cost-effective application of pulsed-field gel electrophoresis to typing of Salmonella enterica Serovar Typhimurium.

Salmonella enterica serovar Typhimurium is frequently isolated from humans and animals. Phage typing is historically the first-line reference typing technique in Europe. It is rapid and convenient for laboratories with appropriate training and experience, and costs of consumables are low. Phage typing and pulsed-field gel electrophoresis (PFGE) were performed on 503 isolates of serovar Typhimurium. Twenty-nine phage types and 53 PFGE patterns were observed. Most isolates of phage types DT104, DT104b, and U310 are not distinguishable from other members of their phage type by PFGE. By contrast, PFGE of isolates of phage types DT193 and U302 shows great heterogeneity. Analysis of experience with PFGE and phage typing can facilitate the selective application of PFGE to maximize the yield of epidemiologically relevant additional information while controlling costs.

Salmonella enterica serovar Kedougou contamination of commercially grown mushrooms.

During investigation of an episode of Salmonella enterica serovar Kedougou contamination of mushrooms, multiple closely related isolates were obtained from mushrooms and mushroom-growing materials. Contamination apparently originated from sugar beet lime, an alkaline material used in mushroom growing. No associated cases of human infection were detected.

Comparison of phenotypic and genotypic characteristics of Salmonella bredeney associated with a poultry-related outbreak of gastroenteritis in Northern Ireland.

OBJECTIVES: To employ a combination of phenotypic and genotypic subspecies typing methods to aid in an epidemiological investigation of an outbreak of Salmonella bredeney involving ten persons. METHODS: Isolates were characterised by employing antibiogram typing, in addition to two genotyping techniques, including pulsed field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD) with two oligonucleotide primers. RESULTS: An outbreak of gastroenteritis associated with S. bredeney (serovar O:4 H:Lv 1,7) occurred in Belfast, Northern Ireland in November 1997. In total, ten cases were confirmed, of which eight had consumed chicken cooked at local butchers and retailed through one of two local bakeries. One of the remaining cases was secondarily infected within her home and the final case had eaten a product other than cooked chicken from one of the bakeries. Food preparation practices were inadequate in one of the bakeries in question and record keeping and possibly cooking procedures were inadequate in the butchers. S. bredeney was isolated from an uncooked chicken supplied to the butchers confirming that improperly cooked chicken was most likely the source of the outbreak. All outbreak clinical isolates were indistinguishable from each other and were similar to the isolate obtained from the uncooked poultry demonstrating that these DNA-based methods were valuable in the molecular characterization of S. bredeney. CONCLUSIONS: This report emphasises the importance and maintenance of an effective hazard analysis critical control point (HACCP) approach to the processing and retailing of foodstuffs containing chicken in order to help eliminate hazards to public health.

Antimicrobial resistance and genetic diversity of Shigella sonnei isolates from western Ireland, an area of low incidence of infection.

Shigella sonnei is a significant cause of gastroenteritis in both developing and industrialized countries. Definition of the diversity and antimicrobial susceptibility of S. sonnei isolates may be helpful in the management of individual cases and outbreaks. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed with 67 isolates of S. sonnei predominantly (n = 59) from three counties in the west of Ireland. Phage typing (n = 17), plasmid profiling (n = 28), and integron analysis (n = 24) were performed with subsets of strains. PFGE typing permitted recognition of two major clusters: PFGE type A (n = 53) and PFGE type B (n = 14). PFGE type A was associated with resistance to ampicillin, streptomycin, and sulfonamides (51 of 53 isolates), and those that were phage typed (n = 6) were phage type 3. PFGE type B was associated with resistance to streptomycin, sulfonamides, tetracycline, and trimethoprim (11 of 14 isolates) and phage type 6 (9 of 11 isolates). Fifteen different plasmid profiles were identified among the 28 isolates analyzed. A class 2 integron was present in all 14 PFGE type B isolates. One of these isolates also contained a class 1 integron and showed a unique variant of the PFGE type B pattern. Sequence analysis of the gene cassette structures contained within these integrons identified distinct open reading frames that encoded determinants of resistance to trimethoprim, streptomycin, and streptothricin. Our data demonstrate two predominant PFGE types among S. sonnei isolates circulating in this region. The limited diversity of the S. sonnei isolates in this region means that detection of isolates indistinguishable by PFGE and according to their antibiograms in two or more patients is not persuasive evidence of a common-source food- or waterborne outbreak. Indistinguishable plasmid profiles in addition to indistinguishable PFGE and antibiogram types may be more suggestive of an epidemiologically relevant link between cases.

Salmonella enterica serotype Bredeney: antimicrobial susceptibility and molecular diversity of isolates from Ireland and Northern Ireland.

Salmonella enterica serotype Bredeney has emerged as the third most commonly identified serotype among human clinical isolates referred to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. A collection of 112 isolates of S. enterica serotype Bredeney collected during the period 1995 to 1999 from animal, food, and human sources from both Ireland and Northern Ireland were studied. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and DNA amplification fingerprinting (DAF) were performed on all isolates. Plasmid profiles were examined on a subset of 33 isolates. A high proportion (74%) of isolates were susceptible to all antimicrobial agents tested. Resistance to both sulfonamide and trimethoprim was observed in 21% of isolates, and resistance to multiple (five) antimicrobial agents was observed in a single isolate (0.9%). Eight different PFGE patterns were obtained, with 87% of isolates grouping as PFGE type A. PFGE type A was predominant in animals, food, and humans. There was good overall concordance between the groups identified by PFGE and DAF. Overall results indicate that most S. enterica serotype Bredeney isolates in Ireland and Northern Ireland from animal and human sources are clonally related.