Duration of Colonisation With Extended-spectrum Beta-lactamase-producing Escherichia Coli: Results of an Open Cohort Study With Dutch Nursing Home Residents (2013 – 2019)

Escherichia coli sequence type ST131 is a recently emerged worldwide pandemic clonal group. Antibiotic resistance, virulence factors or colonisation tness are mentioned among other as possible factors contributing to the worldwide success. In this study, we assessed the duration of rectal ESBL- producing E. coli colonisation in the residents, and compare duration of colonisation for ESBL-ST131 versus ESBL-non-ST131.


Abstract
Background Escherichia coli sequence type ST131 is a recently emerged worldwide pandemic clonal group. Antibiotic resistance, virulence factors or colonisation tness are mentioned among other as possible factors contributing to the worldwide success. In this study, we assessed the duration of rectal ESBL-producing E. coli colonisation in the residents, and compare duration of colonisation for ESBL-ST131 versus ESBLnon-ST131.

Methods
Rectal or faecal samples were obtained from residents of nursing home A between 2013 and 2019 and nursing home B between 2017 and 2019, with repeated point prevalence surveys at intervals of three to six months. Extended-spectrum β-lactamase (ESBL)-producing strains of E. coli were identi ed on selective culture and selective enrichment broth, and examined by antimicrobial susceptibility testing. In nursing home A multilocus sequence typing (MLST) and cluster analyse was performed by respectively O25:ST131-speci c PCR and ampli ed fragment length polymorphism (AFLP). In nursing home B whole genome sequencing data were used to determine MLST and to perform a cluster analyse. Kaplan Meier survival analysis was performed to calculate the median time of rectal colonisation of ESBL-EC with a Log-Rank analysis to test for differences between ESBL-ST131 and ESBL-non-ST131.

Conclusion
Here we found a prolonged colonisation duration of ESBL-ST131 compared to ESBL-non-ST131 in residents of Dutch nursing homes. Prolonged colonisation duration complicates the controlling and ending an ESBL-ST131 outbreak, especially in long stay settings such as nursing homes.

Background
Background/rationale Escherichia coli (E. coli) sequence type (ST) 131 is an extraintestinal pathogenic E. coli (ExPEC) that has emerged recently (1,2), and is nowadays the predominant E. coli lineage among ExPEC isolates worldwide (3). Moreover, ST131 is associated with the worldwide spread of the CTX-M-15 extended spectrum β-lactamase (ESBL) resistance gene (3). ESBL producing ST131 (ESBL-ST131) is a major contributor to hospital-and community-acquired infections, such as urinary tract infection and bloodstream infections (4,5). Whereby ESBL-ST131 infections are most common among elderly and ESBL-ST131 carriage is particularly prevalent in nursing homes and long-term care facilities (6,7). Despite many studies examining the epidemiology of ESBL-ST131, the reason why this clone achieved such considerable success in such a short time span is still unclear. Antibiotic resistance, virulence factors or colonisation tness are mentioned among other as possible factors contributing to this success (3).
In 2016, Overdevest et al. evaluated an ESBL-ST131 outbreak in a Dutch nursing home (nursing home A) between March 2013 and April 2014, whereby six point prevalence surveys were performed at intervals three months by culturing faeces or rectal swabs from all residents (8). The study showed a prolonged colonisation duration of residents with ESBL-ST131, with a median colonisation duration of 13 months compared to two to three months for other ESBL-producing E. coli (ESBL-non-ST131) (p < 0.001).
The point prevalence surveys were continued until June 2019 in nursing home A. In this study, we evaluated the duration of ESBL-producing E. coli (ESBL-EC) colonisation in nursing home A over the period of 6 years (March 2013 to June 2019). In addition, we included a ST131 outbreak in a second Dutch nursing home (nursing home B) from the period March 2017 to June 2019.
The objective of this study was to assess the duration of rectal ESBL-producing E. coli colonisation in residents of two Dutch nursing homes, and compare duration of colonisation for ESBL-ST131 versus ESBL-non-ST131.

Methods
Detection of the ST131 outbreak and setting As part of standard infection control measure, a prevalence survey was performed in the two nursing homes (nursing home A in 2012 (7), nursing home B in 2016) (Fig. 1). Both surveys showed a high prevalence of rectal ESBL colonisation of 20,6% (7) in nursing home A and 13,9% in nursing home B.
Strain typing showed the presence of different clusters of ESBL-ST131, among other smaller clusters and unique strains of other sequences types in both nursing homes. Outbreak measures were implemented, including repetitive prevalence surveys at intervals of three to six months by culturing faeces or rectal swabs from all residents. In June 2019, the prevalence surveys were ended in both nursing homes.
Nursing home A is located on one location consisting of four semi-separate buildings with a total of nine wards. Nursing home B consists of ve different locations with a total of 15 wards. In two wards (on two locations), an ST131 outbreak was detected, which were included in this study. In both nursing homes, wards housed about 20 residents and contained communal areas. Sanitary facilities were shared by several residents each. Nursing staff was dedicated to speci c wards. The locations contained communal recreation and therapy areas where residents from all wards met regularly.

Study design and study population
We conducted an open cohort study with repeated point prevalence surveys from March 2013 to June 2019 in nursing home A and from March 2017 to June 2019 in nursing home B in the Netherlands.
Residents with at least one ESBL-EC positive rectal or faecal swab in a prevalence survey in the study period were eligible for inclusion. Residents acquiring colonisation in the nal prevalence survey were excluded.

De nitions
Rectal colonization with ESBL-EC was de ned as detection of ESBL-EC in at least one rectal swab or faecal sample. A resident was considered no longer colonised (loss of colonisation) when one rectal swab or stool sample no longer yielded ESBL-EC or when strain typing showed a different cluster or sequence type than found in the previous ESBL-EC positive culture of the resident. Residents were only included in the study once.

Variables
Data concerning gender, date of birth, day of discharge from nursing home or day of death were obtained from the nursing homes records.

Detection of ESBL-producing E. coli
The intended sampling schedule consisted of a quarterly screening in all residents of the wards involved, in nursing home A and B. Rectal colonisation of ESBL-EC was determined by culture of rectal or faecal samples (Eswab, Copan, Italy). Swabs were inoculated on extended-spectrum β-lactamase screening agar (EbSA) plate (AlphaOmega,'s-Gravenhage, Netherlands) and 5% Sheep blood agar (growth control). The remaining Eswab uid was transferred in 5 mL tryptic soy broth containing cefotaxime (0.25 mg/L) and vancomycin (8 mg/L) (TSB-VC). After 18-24 hours incubation (35-37 °C), the TSB-VC was subcultured on an EbSA plate. For all Gram-negative rods growing on the EbSA, species identi cation and susceptibility testing was performed by MALDI-TOF (bioMérieux, Marcy l'Etoile, France) and VITEK 2 (bioMérieux, Marcy l'Etoile, France), respectively. Phenotypic ESBL production was con rmed by double disk method (9) Genotyping and strain typing Nursing home A All phenotypically con rmed ESBL-EC underwent an O25:ST131-speci c PCR (10). ESBL genotyping was performed using a micro-array (CheckPoints, Wageningen, the Netherlands) (11,12) and strain typing by using ampli ed fragment length polymorphism (AFLP) (13). An AFLP cluster was assigned based on both visual and computerised interpretation of AFLP patterns.
Genotyping and strain typing were performed for the rst ESBL-EC from each resident and for and subsequent ESBL-EC strains that were not similar to the rst strain. Similarity was de ned as identical species, identical phylogroup and O25:ST131 status and absence of major differences in susceptibility (susceptible vs. resistant) for the all antibiotics tested.
Nursing home B All phenotypically con rmed ESBL-EC were sent to the University Medical Center Groningen, The Netherlands, for whole-genome sequencing (WGS) and subsequent downstream data analysis multilocus sequence typing (MLST), Whole-genome MLST (wgMLST) (core and accessory genome) and resistome analysis was performed at Microvida, location Amphia, The Netherlands. Genomic DNA was extracted using the Ultraclean Microbial DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, US) following the manufacturer's instructions. The DNA library was prepared using the Nextera XT v2 kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions and then run on the MiSeq (Illumina) for generating paired-end 250-bp or 300-bp reads (Table 1). De novo assembly was performed by CLC Genomics Workbench version 10.0.3, 10.1.1, 11.0, 11.0.1 or 12.0 (QIAGEN, Hilden, Germany) ( Table 1) after quality trimming (Qs ≥ 20) with optimal word sizes (Kmer size 30). Assembled genomes were typed using MLST and wgMLST with SeqSphere version 6.0.2 (Ridom GmbH, Münster, Germany). Clusters were de ned using wgMLST% identity via distance matrix, with cluster cut-off criteria described by Kluytmans et al. (14). Assembled genomes were uploaded to the webtool ABRicate on the Galaxy platform version 1.0.1 (https://usegalaxy.eu) using the Res nder database (last update September 10, 2019) for identifying the acquired resistance genes with default settings (minimal coverage 60%; %ID75%). For each resident, the time of rectal colonisation of ESBL-EC was calculated as the time from the rst ESBL-EC positive sample until the last ESBL-EC positive sample plus the half-time between the last ESBL-EC positive culture and the rst ESBL-EC negative culture (Fig. 2a) or the rst ESBL-EC positive culture with a different sequence type or cluster type (Fig. 2b). Both situations were labelled as an 'event' in the Kaplan Meier survival analysis.
In residents whose last sample was still ESBL-EC positive (no loss of colonisation) at the end of the study period, the time of rectal colonisation of ESBL-E. coli was calculated as the time from the rst ESBL-EC positive sample until the last ESBL-EC positive sample (Fig. 2c). Residents who died or were discharged before the of the study, the time of rectal colonisation of ESBL-E. coli was calculated as the time from the rst ESBL-EC positive sample until time of death or discharge (Fig. 2d). These cases were labelled as 'censored' in the Kaplan Meier survival analysis.

Results
Between March 2013 and June 2019, 23 point prevalence surveys were performed in nursing home A and nine point prevalence surveys in nursing home B at intervals of three months to six months by culturing faecal or rectal swabs from all residents (n = 1059 unique residents). In total, 155 residents had at least one ESBL-EC positive sample in a prevalence survey between (Fig. 3). Of these, eleven residents were excluded from the study because O25:ST131-speci c PCR was not performed (n = 5), result of the O25:ST131-speci c PCR or WGS MLST was inconclusive (n = 2), resident acquired ESBL-EC in the nal prevalence survey (n = 6) or the resident carried an ESBL-ST131 and ESBL-non-ST131 in the rst sample (n = 1). For one resident, wgMLST cluster analysis showed 18 different ST131 strains in nine rectal samples of the resident. Further analysis of the WGS data showed that the hypermutation of these ST131 strains were due to a defect in mutA gene which involves in the mismatch repair mechanism (15). Consequently, determining duration of colonisation at strain level was not possible and the resident was excluded from the study.
Additionally, a further analysis was based on the strati cation of the ESBL-ST131 and ESBL-non-ST131 groups for age, gender and nursing home. In the subgroup ST131, median colonisation length was signi cantly longer in female than in males: 25,6 months [95% CI, 0,6-50,7] versus 8,1 months [95%CI, 5,9-10,3] (p = 0,013) (S3-5). For age and nursing home, no statistical differences were found in the subgroup analysis. , with a median of 13 months compared to two to three months for other ESBL-producing E. coli (ESBL-non-ST131) (p < 0.001) (8). In this study, we evaluated the duration of rectal ESBL-producing E. coli colonisation in residents of nursing home A for a study period of six years (2013-2019) and included a second Dutch nursing home (nursing home B) with a study period of two years. (2017-2019). In concordance with Overdevest et al. we found a prolonged colonisation of residents with ESBL-ST131, with a median of 13 months compared to eight months for ESBL-non-ST131 (p = 0,028). Remarkably, in the subgroup ST131 the median colonisation length was signi cantly longer in female than in males: 25,6 months versus 8,1 months (p = 0,013).

Discussion
On a resident level, prolonged colonisation is a risk for the reason that colonisation can proceed to (extraintestinal) infections (16) for an extended time, and extraintestinal infections due to Escherichia coli cause considerable morbidity, mortality, and increased health care costs (17).
At the institutional level, prolonged colonisation is a risk as colonised residents can contribute to transmission for an extended times. And once ESBL-ST131 is introduced and spread in a nursing home, it will take a lot of effort and time for ESBL-ST131 to disappear from a nursing home setting partly because of the prolonged duration of colonisation (8).
There  (25). These traits (improved bio lm production and high metabolic potential) probably enhance the ability of ST131 to establish and maintain intestinal colonisation (3,26).
The large difference of median colonisation length between female and male (25,6 months versus 8,1 months) in the subgroup ST131 was an unexpected nding. This nding could not explained by differences in age distribution with in the subgroup, nor by the number of residents who died during the study period. (S3). A possible explanation could be a difference in the presence of known risk factors for (prolonged) ESBL colonisation between the sexes, such as antibiotic use, proton pump inhibitor use or variables associated with higher need for care (18,27). Unfortunately, such clinical information was not acquired in this study. Further studies into this observed difference are warranted.
Another limitation of this study is the setting. The study was performed in two Dutch nursing homes in an outbreak situation, which may reduce generalizability for other settings and/or patient populations. The major strength of our study includes the standardised cultures taken at de ned intervals and most important the long follow-up period up to six years in nursing home A and 2 years in nursing home B.

Conclusion
Understanding the dynamics of ESBL-ST131 in residents in nursing homes is crucial to identify effective infection control measures. Here we found a prolonged colonisation duration of ESBL-ST131 compared to ESBL-non-ST131. A consequence is that once ESBL-ST131 is introduced and spread in a nursing home, it will take a lot of effort and time for ESBL-ST131 to disappear from the setting. More stringent control measures for ESBL-ST131 may therefore be warranted. Availability of data and materials

List Of Abbreviations
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests Funding None Authors' contributions VW, TdG, YH, LK, AM, BvdW and JK performed the investigation and management of the outbreak. VW and JK did the data analysis. VW wrote the rst draft of the manuscript, TdG, YH, LK, AM, BvdW and JK reviewed, provided critical feedback and contributed to subsequent draft. All authors approved the nal version for publication. Figure 1 Overview of the outbreak detection and subsequent prevalence studies in nursing home A and nursing home B.