Molecular Medicine Israel

Within-host genetic diversity of extended-spectrum beta-lactamase-producing Enterobacterales in long-term colonized patients


Despite recognition of the immediate impact of infections caused by extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) on human health, essential aspects of their molecular epidemiology remain under-investigated. This includes knowledge on the potential of a particular strain to persist in a host, mutational events during colonization, and the genetic diversity in individual patients over time. To investigate long-term genetic diversity of colonizing and infecting ESBL-Klebsiella pneumoniae species complex and ESBL-Escherichia coli in individual patients over time, we performed a ten-year longitudinal retrospective study and extracted clinical and microbiological data from electronic health records. In this investigation, 76 ESBL-K. pneumoniae species complex and 284 ESBL-E. coli isolates were recovered from 19 and 61 patients. Strain persistence was detected in all patients colonized with ESBL-K. pneumoniae species complex, and 83.6% of patients colonized with ESBL-E. coli. We frequently observed isolates of the same strain recovered from different body sites associated with either colonization or infection. Antimicrobial resistance genes, plasmid replicons, and whole ESBL-plasmids were shared between isolates regardless of chromosomal relatedness. Our study suggests that patients colonized with ESBL-producers may act as durable reservoirs for ongoing transmission of ESBLs, and that they are at prolonged risk of recurrent infection with colonizing strains.


Extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) are considered a critical threat by public health authorities, such as the Centers for Disease Prevention and Control (CDC) and the World Health Organization (WHO)1,2. Infections with ESBL-PE are associated with excess morbidity, mortality, and higher health-care costs. Despite this recognition of the immediate impact on human health, important aspects regarding the molecular epidemiology of ESBL-PE remain largely under investigated. In particular, there is a lack of knowledge regarding the potential of a particular strain to persist in a host over long time periods, the number of mutational events occurring within a host during colonization, and the genetic diversity of colonizing and infecting isolates within an individual patient and over time. Such knowledge is critical for a deeper understanding of the ESBL-PE epidemiology requiring data on reservoirs and transmission chains, their sustainability and thus their potential to persist over time. Further, investigation of bacterial in-host mutation events may provide important knowledge on the host-pathogen interactions required to establish successful colonization and subsequently infection. So far, ESBL-PE colonization has been mainly studied in presumably “healthy” returning travelers or patients with limited follow-up periods usually not longer than 24 months3,4,5,6. In addition, these studies mostly lack detailed whole-genome analyses3,4,7.

At our institution, patients colonized with ESBL-PE are routinely screened at different body sites upon each admission and at least one ESBL-PE isolate recovered by routine clinical sampling is systematically collected. In this work, we investigate the within-host genetic diversity of colonizing and infecting ESBL-Klebsiella pneumoniae species complex and ESBL-Escherichia coli isolates and their plasmids by sequencing these isolates, collected over a 10-year period from different screening and clinical samples.



From 2008–2018, 495 known ESBL-carriers were prospectively screened by performance of rectal swabs at each visit. Of these, 73 consecutive patients with isolation of at least two ESBL-PE belonging to the same species (K. pneumoniae or E. coli) from two consecutive rectal swabs were included in this study. Twelve of them were colonized with ESBL-K. pneumoniae species complex, 54 with ESBL-E. coli, and seven patients were colonized with both ESBL-PE. The Supplementary Table S1 summarizes the baseline characteristics of all the patients included in this study, stratified by species.

Bacterial isolates

During the 10-year study period, 360 ESBL-PE isolates were recovered (76 K. pneumoniae and 284 E. coli) (Supplementary Fig. S1). Among isolates initially classified as K. pneumoniae by conventional methods, 70 isolates were confirmed as belonging to K. pneumoniae sensu stricto, five were identified as K. quasipneumoniae (K. quasipneumoniae subsp. quasipneumoniae n = 3 and K. quasipneumoniae subsp. similipneumoniae n = 2) and one as K. variicola subsp. variicola by sequencing. Virulence analyses done for all K. pneumoniae species complex isolates revealed no hypervirulent strains. A detailed summary of the Kleborate results is shown in the Supplementary Data S3.

Most isolates were recovered from screening samples (315/360, 87.5%), the rest (45/360, 12.5%) were recovered from samples collected due to suspected infection (4/76, 5.6% of all K. pneumoniae species complex and 41/284, 14.4% of all E. coli isolates). The majority of isolates was recovered from rectal swabs (237/360, 65.8%), while the remaining isolates (123/360, 34.2%) were obtained from other body sites.

A median of four isolates per patient (IQR 3–5) were collected (Table 1). The maximum number of isolates collected per patient was eight and 19 for K. pneumoniae species complex and E. coli (Table 1, Supplementary Fig. S2).

Bacterial diversity and multi-locus sequence typing results

The overall sequence type (ST) diversity per species was high, with a Simpson’s diversity index of 0.929 for K. pneumoniae species complex and 0.803 for E. coli. Isolates were allocated to 20 different STs of K. pneumoniae species complex and 38 different STs of E. coli. Among the 20 different STs of K. pneumoniae species complex, ST48 (n = 12), ST45 (n = 10), ST307 (n = 8), ST985 (n = 7) and ST394 (n = 5) were the five most abundant ones. In E. coli, ST131 (n = 120), ST648 (n = 23), ST10 (n = 18), ST405 (n = 15) and ST362 (n = 14) were the most abundant ones. The full list of STs identified is provided in the Supplementary Data S1. According to the cgMLST analyses, 93.4% of K. pneumoniae species complex isolates and 80.2% of E. coli isolates were grouped into 17 and 53 clusters, respectively.

cgMLST clusters and strain persistence within the same patient

In 84.9% of patients (62/73) only one cluster of ESBL-PE was identified (Fig. 1). In one patient colonized with K. pneumoniae species complex and five patients colonized with E. coli, two different clusters were detected (Fig. 1).

Fifteen and 28 patients were colonized with only one strain within ESBL-K. pneumoniae species complex and ESBL-E. coli isolates, respectively (43/73, 58.9%). The rest of the patients (4 colonized with K. pneumoniae species complex and 33 patients colonized with E. coli) harbored more than one bacterial strain of each species (Fig. 1). One patient (Pat02) carried six different strains belonging to different species: E. coli (two strains from ST648), K. pneumoniae (two strains from ST20 and ST422), K. variicola (one strain), and K. quasipneumoniae (one strain) during an eight-year time frame. Detection of >1 ESBL-PE species (K. quasipneumoniae and E. coli) at the same hospital admission occurred once.

Persistent carriage of the same strain was detected for 100% of patients colonized with K. pneumoniae species complex and 83.6% of patients colonized with E. coli. For patients colonized with K. pneumoniae species complex the median time between collection of the first and the last isolate was 445 days (IQR 210.5–909); for patients colonized with E. coli, 456 days (IQR 258–1230 days) (Table 1, Fig. 2A). Colonization with the same strain persisted for a median of 231 days (IQR 106–535) and 332 days (IQR 170–780) for K. pneumoniae species complex and E. coli, respectively. Persistent colonization by the same ESBL-E. coli and/or K. pneumoniae strain lasted for at least one, three and five years in 20.5%, 6.8% and 5.5% of patients, respectively. The longest observed strain persistence was 1704 days for K. pneumoniae (Fig. 2B) and 3387 days for E. coli (Supplementary Table S2, Fig. 2B)….

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