Genomic surveillance of antimicrobial resistant bacterial colonisation and infection in intensive care patients

Healthcare associated infections (HAI) result in considerable morbidity and mortality, with the total burden in high income countries exceeding that of influenza, tuberculosis and other major communicable diseases combined [1]. The emergence and spread of antimicrobial resistant (AMR) organisms, including difficult-to-treat multi-drug resistant (MDR) strains, has further exacerbated this problem [2], and as a consequence several MDR healthcare-associated bacterial pathogens are now recognised by the World Health Organization (WHO) as urgent threats to public health [3]. Among the WHO’s top priorities are carbapenem-resistant Gram-negatives (GNs, specifically Enterobacteriaceae, Acinetobacter baumannii and Pseudomonas aeruginosa) as well as vancomycin-resistant enterococci (VRE).

In Australia, it is estimated that one in ten hospitalised patients suffers an HAI, and the most common MDR organisms are VRE, methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase (ESBL) producing Enterobacteriaceae [4]. Fortunately carbapenem-resistance has so far remained rare, although the prevalence has been slowly increasing [5]. Notably, Australia suffers a particularly high rate of vancomycin resistance among enterococcal infections (~ 45% of Enterococcus faecium bacteremias [5]), primarily due to the endemic spread of healthcare-associated E. faecium sequence types (STs) 17, ST80 and ST796 [5‐8].

Asymptomatic gut colonisation is considered a key risk factor for enterococcal, Enterobacteriaceae and other GN infections [9‐13] and in the case of VRE has been associated with longer ICU stay and treatment costs [12, 14]. A number of hospitals have implemented rectal screening programs to identify patients colonised with VRE and/or carbapenemase-producing Enterobacteriaceae in high-risk wards such as intensive care units (ICUs), oncology and hematology wards [7, 8, 15, 16]. However, few facilities conduct routine screening for ESBL-Enterobacteriaceae or other high-risk carbapenem-susceptible GNs in part due to a lack of evidence to inform the interpretation of these results and to appropriately target infection control measures. For example, only a small number of studies have investigated the risks associated with ESBL-Enterobacteriaceae colonisation at a species-specific level [9, 11, 12, 17‐20], but there is emerging evidence to suggest important variation, both in terms of the risk of infection and the risk of transmission in the hospital setting. We propose that a better understanding of these risks, as well as the broader colonisation and infection dynamics of AMR organisms in these settings, will facilitate better targeting of infection prevention and control practices.

Here we report a genome-resolved snapshot of high-risk AMR bacteria from rectal swabs and clinical specimens collected in an Australian ICU over a three-month period. We focus on GN organisms resistant to third-generation cephalosporins (including ESBL-Enterobacteriaceae) in addition to VRE. We leverage the genome data to perform high-resolution analysis of these bacterial populations to; i) compare infecting and colonising organisms isolated form the same patient; ii) characterise population diversity at the strain level; and iii) identify putative nosocomial transmission clusters.

During the study period there were 716 patients admitted to the ICU, 31 (4.3%) patients had one or more 3GCR-GN infections (n = 41 isolates), and two (0.3%) had VRE infections (n = 2 isolates, both E. faecium, Fig. 1). Of 430 patients eligible for participation in rectal screening, 66 declined to participate and 311 contributed one or more rectal swabs (72.3% of eligible non-refusers). The majority of participants were swabbed within the first 2 days of ICU admission (baseline screening swabs, n = 287, 92.3%), including 79 with ≥1 additional follow-up swab (Fig. 1). Participants with baseline swabs were 64.5% male (n = 185), aged 18–93 years (median 57 years; IQR, 44–71 years, Supplementary Table 2, Additional File 1), and the majority (n = 218, 76.0%) were known to have had recent healthcare exposure prior to ICU admission (surgical procedure within the last 30 days, transferred from another ward with first admission > 2 days prior, or transferred from another hospital).

Third generation cephalosporin-resistant (3GCR) GN organisms were isolated from baseline screening swabs of 50 patients (17.4%, n = 56 isolates), while VRE were isolated from 24 (8.4%, n = 24 E. faecium isolates, no E. faecalis were identified; Supplementary Figure 1, Additional File 1). Co-colonisation with 3GCR-GN and VRE was identified in 12 patients (4.2%), indicating a significant association between these organisms (OR 5.6, 95% CI 2.2–15.5, p = 0.0001 using Fisher’s Exact Test). There were no significant differences in colonisation rates between males and females, and no association between age and colonisation status, with the exception of VRE in females (median age 73.5 years amongst carriers versus 55.5 years amongst non-carriers, p = 0.04 using Wilcoxon Rank Sum test; Fig. 1). Neither surgery within the last 30 days, recent healthcare exposure (defined as above), or antibiotic treatment within the last 7 days, were significantly associated with 3GCR-GN or VRE colonisation at baseline (using univariate and multivariate logistic regression, with age and sex as covariates; Supplementary Table 3, Additional File 1).

A total of 138 rectal swabs were collected from 103 patients between 3 days and 12 weeks after ICU admission (Supplementary Table 4, Supplementary Figure 2, Additional File 1). Twenty-five swabs (18.1%) from 18 patients (17.5%) were positive for ≥1 3GCR-GN organism (n = 30 isolates); 15 swabs (10.9%) from ten patients (9.7%) were positive for VRE (n = 15 isolates). Notably, 3GCR-GN organisms were cultured from 15/61 patients (24.6%) who were culture-negative for 3GCR-GN colonisation at baseline, and VRE were cultured from 13/75 (17.3%) patients who were culture-negative for VRE colonisation at baseline, consistent with acquisition and/or overgrowth of the organisms during ICU stay.

Among the 127 3GCR-GN and 41 VRE isolates cultured from ICU patients, 112 (88.2%) and 41 (100%) respectively were successfully revived and sequenced (Supplementary Methods, Supplementary Figure 1, Additional File 1). We combined clinical and genome data to identify distinct colonisation and infection episodes, defined as unique combinations of species and ST (derived from whole-genome sequences (WGS)), per patient and specimen type. This identified 33 3GCR-GN and two VRE infection episodes (Table 1). The latter included one urinary tract infection for which the patient was prescribed amoxycillin (as recommended for treatment of VRE in the urinary tract, where a high concentration of amoxycillin can be achieved) and one respiratory infection treated with teicoplanin. 3GCR-GN infections were mainly respiratory (n = 19, 57.6%), wound (n = 5, 15.2%) and bloodstream (n = 4, 12.1%) infections, and the most common agents were Escherichia coli (10 episodes, 30.3%) and Klebsiella pneumoniae (6 episodes, 18.2%; Table 1, Fig. 2). E. coli was also the most common 3GCR-GN gut coloniser (36/62 3GCR-GN colonisation episodes, 58%), followed by Enterobacter hormaechei, K. pneumoniae, Klebsiella aerogenes, and Klebsiella oxytoca (four episodes, 6.5%, each, Table 1). Thirty-three unique VRE colonisation episodes were identified. Overall, 88 patients were colonised and/or infected with 3GCR-GN and/or VRE. Most (62, 70.5%) experienced a single episode of either 3GCR-GN (n = 61) or VRE (n = 1), however 17 patients (20.2%) had two episodes (16 patients with two different species/STs, 13 with one VRE and one 3GCR-GN species) and nine patients (10.1%) had ≥3 episodes (seven with ≥3 different species/STs, including five with VRE).

The majority of 3GCR-GN colonisation and infection isolates were predicted to be MDR (see Methods): n = 44/61 (72%) distinct colonisation episodes and n = 18/29 (62%) distinct infection episodes. MDR was particularly common among E. coli (n = 44 episodes, 96%), K. pneumoniae (n = 9, 90%) and Serratia marcescens (n = 3, 100%). WGS identified acquired ESBL genes in 20 representative 3GCR-GN colonisation (32.3%) and 12 representative 3GCR-GN infection (36.4%) isolates. These were mostly MDR K. pneumoniae carrying bla_CTX-M-15 (n = 8) or MDR E. coli carrying diverse bla_CTX-M genes (n = 19, including n = 6 bla_CTX-M-14, n = 5 bla_CTX-M-15, n = 3 bla_CTX-M-27, n = 3 bla_CTX-M-62, n = 2 bla_{CTX-M-24-like}). Carbapenemase genes were rare: two patients had wound or tissue infections with K. pneumoniae ST231 carrying bla_OXA-48, one of whom was also colonised by K. pneumoniae ST231 with bla_OXA-48. A third patient was colonised with S. marcescens carrying bla_IMP-4. One of the patients with K. pneumoniae ST231 wound / tissue infections received a course of meropenem (this patient also had 3GCR P. aeruginosa and 3GCR Burkholderia cenocepacia infections), while the second received meropenem, ciprofloxacin and tigecycline. The S. marcescens rectal colonisation episode was not treated.

Most VRE isolates representing distinct episodes (n = 32, 91%) carried the vanB vancomycin resistance operon and the remainder (n = 3, 9%) the vanA operon, which confers resistance to vancomycin plus teicoplanin [32]. Additionally, 32 of these isolates (91%) were predicted to be MDR due to the presence of genes conferring resistance to tetracyclines and/or high-level resistance to gentamicin, streptomycin and/or streptogramins.

We assessed species-specific infection rates amongst patients testing culture-positive at baseline screening, versus those testing culture-negative, for all species with ≥2 infections identified amongst patients with baseline screening data (E. coli, K. pneumoniae, P. aeruginosa). Infection rates were higher amongst baseline-culture-positive patients, although the difference was statistically significant only for K. pneumoniae and P. aeruginosa (Table 1). There were few patients for whom WGS data were available for isolates of the same species detected in baseline screening and infections (two patients for each species). For n = 2/2 such cases with K. pneumoniae (ST231, ST323) and n = 1/2 with P. aeruginosa (ST357), the WGS data confirmed the infections were caused by the colonising strain (0–7 pairwise SNVs), whereas both E. coli infections were caused by different strains to those detected on baseline screening swabs (different STs; Table 1, Supplementary Results, Additional File 1). A further two patients tested culture-negative on baseline screening, but subsequently tested positive on follow-up screening for the same strain that was isolated from their diagnostic specimens (K. pneumoniae ST323, E. coli ST393; Supplementary Results, Additional File 1).

Finally, we assessed strain level diversity and evidence for nosocomial transmission using WGS and patient data. The isolates were diverse, with 62 STs identified amongst 3GCR-GN (24 amongst E. coli alone, including n = 6 ST131 episodes, n = 4 ST10 and n = 3 episodes each of ST38, ST69, ST176, ST648 and ST963) and 8 amongst VRE (including n = 14 ST796, n = 10 ST17 and n = 5 ST203 episodes). Most 3GCR-GN STs (n = 48, 77%) were unique to a single patient; however, ten E. coli STs (42%), two K. pneumoniae (40%), one K. oyxtoca (33%), one Enterobacter hormachaei (17%) and five VRE STs (63%) were found in multiple patients (Fig. 3a). We defined probable strain transmission events on the basis of chromosomal SNVs (see Methods, Fig. 3b-c). Using a threshold of ≤20 SNVs for transmission of 3GCR-GN (based on recent studies [10, 15, 16, 33]) we identified six putative 3GCR-GN transmission clusters each involving 2–3 patients (maximum 8 SNVs, Table 2). With a single exception, all patients within clusters were epidemiologically linked (overlapping ICU stays or ≤ 14 days between stays). Assuming one patient in each epidemiologically linked cluster was the index patient, the data suggest only six (6.3%) 3GCR-GN episodes resulted from recent nosocomial transmission (n = 2, 6.1% of infections; n = 4, 6.5% of colonisation). We also identified K. pneumoniae ST323 isolates from two patients that differed by 45–47 SNVs, notably many fewer than the majority of comparisons (Fig. 3a) and within the range expected for K. pneumoniae subject to wider circulation in the healthcare network rather than those subject to community-transmission [33]. Consistent with this, we have previously shown that K. pneumoniae ST323 were circulating more broadly within the hospital during the study period [34].

Interpretation of VRE pairwise SNVs is more complex because several healthcare-associated lineages are known to be circulating in Melbourne and the regional hospital network. Consequently, it is not uncommon to find near-identical isolates in patients from different wards, hospitals or cities without epidemiological links [7, 8, 15]. We therefore used a more conservative threshold of ≤3 SNVs to define putative recent VRE transmissions (based on empirical distribution, see Supplementary Figure 3), which identified four clusters of 2–7 patients each (Table 2). Two clusters were ST17 (both epidemiologically linked clusters, separated by ≥2900 SNVs) and two were the locally emerged ST796 [35, 36] (clusters separated by 4–6 SNVs, one was epidemiologically linked). Assuming one patient in each epidemiologically linked cluster is the index patient, we estimate eight (28.6%) VRE episodes resulted from recent transmission (n = 1 infection and n = 7, 21.2% of colonisation episodes). This should be considered a conservative estimate considering the difficulty in distinguishing clusters, particularly for ST796. Nevertheless, the transmission burden is significantly greater than that estimated for 3GCR-GN organisms (OR = 4.3, 95% CI 1.2–16.6, p = 0.02 using Fisher’s Exact test).

We have presented a holistic investigation of 3GCR-GN and VRE colonising and infecting patients in an Australian ICU over a three-month duration. Approximately 17% patients were colonised by at least one 3GCR-GN at ICU admission. Just under 40% of these isolates were detected as ESBL-Enterobacteriaceae, hence our data are comparable to reports from Europe where 7–13% of ICU-admitted patients were colonised with these organisms [17, 19, 37], and indicate a lower prevalence of ESBL-Enterobacteriaceae colonisation than reported among patients admitted to ICUs in China (32%) [38] and Thailand (62%) [39]. Additionally, 9% of patients in our study were colonised with VRE at ICU admission; a similar prevalence to that reported recently for patients in a hospital network in Singapore [40], higher than that reported for patients admitted to an ICU in Sri Lanka (2%) [41], and lower than reported in Brazil (15%) [42] and India (31%) [43]. Notably, the VRE colonisation prevalence was lower than the 17.5% (95% CI 13.7–21.9) indicated in a previous point-prevalence survey of patients in our hospital [44].

E. coli was the dominant colonising and infecting organism, and has been previously reported as the most common cause of GN blood-stream infections in Australia [5]. These findings are consistent with the notion of colonisation as a prerequisite for disease and the logic of scales, whereby the most common colonisers have more opportunities to cause infection and therefore contribute the greatest burden of disease. Our data indicated a higher prevalence of infections among patients colonised at admission with 3GCR E. coli, K. pneumoniae or P. aeruginosa than those who were not colonised. However, there were differences in the rate of infection; just 6% for E. coli, versus 100% (n = 2/2 each) for K. pneumoniae and P. aeruginosa. While these data must be interpreted with caution due to the small sample size, we note that the trends are consistent with previous reports [9, 17] including a large study of 2386 patients colonised by ESBL-K. pneumoniae or ESBL-E. coli, which found that the former were twice as likely as the latter to develop an infection with the same organism (6.8% versus 3.2% [9]).

At the time of this study our hospital infection prevention and control (IPC) policy stated that all patients known to have a clinical VRE and/or carbapenemase-producing Enterobacteriaceae (metallo-beta-lactamases only) infection or colonisation were isolated and subject to contact precautions during their hospital admission. Nevertheless, our WGS analysis identified four putative VRE transmission clusters (including three that were epidemiologically linked) and indicated that 21.2% of VRE episodes were attributable to transmission. In contrast, we estimated that just 6.3% of 3GCR-GN episodes were attributable to transmission (21.2% VRE vs 6.3% 3GCR-GN, OR 4.3, p = 0.0209), resulting from six putative 3GCR-GN transmission clusters (all epidemiologically linked). However, no specific IPC policies were applied for patients known to be colonised or infected with 3GCR-GN organisms beyond standard care.

The putative VRE transmission clusters involved ST17 and ST796, which are considered healthcare-adapted lineages and are widely distributed in Australia [5‐8, 36, 45]. While four of the six 3GCR-GN transmission clusters involved E. coli, these included just four of 24 distinct E. coli STs, and did not include the globally distributed ST131, ST10 or ST38. This is consistent with a recent report showing that these E. coli STs were commonly identified from patients in other Melbourne hospitals, but rarely associated with nosocomial transmission [15]. Hence our data adds to the growing evidence base [15, 18‐20, 46] that ESBL-E. coli are mainly spread in the community setting.

While the risk of ESBL-E. coli transmission in hospitals may be low, there is emerging evidence that the risk may be 2–4 times greater for other ESBL-Enterobacteriaceae such as K. pneumoniae and Enterobacter sp. [19, 20]. In our study, 2/11 (18.2%) ESBL-K. pneumoniae episodes were attributed to recent transmission, with one additional episode attributed to broader transmission within the hospital as reported previously [34]; however, a larger sample size is required to make a definitive comparison.