Longitudinal genomics reveals carbapenem-resistant Acinetobacter baumannii population changes with emergence of highly resistant ST164 clone

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Nature

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Longitudinal genomics reveals carbapenemresistant Acinetobacter baumannii
population changes with emergence of highly resistant ST164 clone

Carbapenem-resistant Acinetobacter baumannii is an important nosocomial pathogen that can cause outbreaks. In 2019, these authors conducted a genomic surveillance study of A baumannii in an intensive care unit in China, and here they report findings from

A. baumannii infections were rare despite a high abundance in patients and bed units

A total of 131 patients (79 male; 52 female; median age 68 years; interquartile range [IQR] = 55.5–77.5) were sampled over the 3-month study period (Table 1). The median length of patient stay, from admission to discharge or the end of the study, was 10 days (IQR: 5–21.5 days). Most patients (53.4%) were in the ICU for ≤10 days, but 17.1% were present for >31 days. Moreover, the median length of ICU stay of A. baumannii-infected patients (clinical isolative positive) was 28.5 days (IQR: 24–42.3 days). The median length of other ICU patients was 9 days (IQR: 5–16 days). The length of stay (LoS) of A. baumannii-infected patients was significantly longer than other patients in the ICU. Samples were taken each Tuesday, and patients were screened within the first 48 h after admission when possible. “Bed units”, defined as the environmental sites of each bed and its associated equipment, were sampled on 363/364 (99.7%) planned sampling occasions, as one patient was undergoing bedside surgery on a single planned sampling occasion. Patients were sampled on 303/346 (87.6%) planned occasions.

Table 1 Clinical data of patients who were in the ICU during this study

Full size table

A total of 5341 samples were collected from patients and environmental sites. A. baumannii was isolated from 505 samples: 382/4450 (8.6%) environmental samples and 123/891 (13.8%) patient samples. Bed units yielded more environmental isolates (374/4317, 8.7%) than communal areas outside bed units (8/133 samples, 6.0%). Within bed units, ventilators (56/281 samples, 19.9%) and ventilator shelves (40/275 samples, 14.6%) yielded the most A. baumannii. Of the 131 patients screened, 41.2% (54/131) were A. baumannii-positive on at least one sampling occasion, with most of the positive samples originating from nasogastric tube (33/174, 19.0%) or oral (40/220, 18.2%) swabs, followed by rectal swabs (38/273, 13.9%), nasojejunal tube (4/49, 8.2%), endotracheal tube (5/94, 5.3%) and tracheostomy tube (3/81, 3.7%). A set of 13 isolates obtained from diagnostic clinical specimens were collected from 10 patients over the study period; one from an abdominal excision exudate and 12 from sputum.

Most ICU A. baumannii were carbapenem-resistant

Over the study period, 19.1% (25/131) of ICU patients were treated with carbapenems imipenem, meropenem, or ertapenem. MIC testing of all 518 A. baumannii in this collection revealed that the rates of resistance to both imipenem and meropenem were 80.9% (419/518) (Fig. S4). A larger proportion of patient isolates (116/123; 94.3%) were CRAB than environmental isolates (303/382; 79.3%). However, the isolation rate of CRAB clinical strains of patients decreased from 8.6% (2019) to 7.6% (2021). The β-lactam/β-lactamase inhibitor combinations cefoperazone/sulbactam and piperacillin/tazobactam were administered to 14.5% (19/131) and 40.5% (53/131) of patients, respectively. The resistance rates to these combinations were 80.3% (416/518) for cefoperazone-sulbactam (1:1) and 81.1% (420/518) for cefoperazone-sulbactam (2:1). Resistance rates to sulbactam alone, ciprofloxacin, and amikacin were 80.9% (419/518), 80.9% (419/518), and 23.0% (119/518), respectively. All isolates were sensitive to colistin and tigecycline.

To quantify levels of carbapenem resistance in the CRAB populations from before and after the intervention period, we compared the imipenem MICs that would be sufficient to inhibit 50% (MIC50) and 90% (MIC90) of all isolates in each collection. For the 419 CRAB isolated in this study, the MIC50 and MIC90 to imipenem were 64 and 128 mg/L, respectively. The imipenem MIC50/MIC90 values for the 551 CRAB isolated in our first study were 32 and 64 mg/L, respectively. This data reflects an increase in carbapenem resistance levels in this ICU between 2019 and 2021. However, total sensitivity to colistin and tigecycline remained consistent across study periods.

Whole-genome sequencing revealed shifts in the circulating CRAB population

All (n = 518) A. baumannii isolates were whole-genome sequenced (Supplementary Data 1). Genome sequence data revealed that the population included representatives of 36 different sequence types according to the Pasteur MLST scheme (Fig. 1A). Most of the diversity was seen amongst the carbapenem-sensitive population, which included isolates from 34 different STs, 20 of which were represented by a single isolate each. Only three of these STs were represented by ten or more isolates: ST240, ST40, and ST221. The carbapenem-resistant population was comprised of just two sequence types: ST2/GC2 (213 isolates; 50.8%) and ST164 (206 isolates; 49.2%). The presence of ST164, accounting for approximately half of the CRAB isolates in this study, represents a major shift from 2019, where the CRAB population was dominated by GC2 (548/551; 99.5%) (Fig. 1A). ST164 isolates were obtained from clinical specimens in this ICU earlier in 2021, and this ST has recently been described as a new international clone (IC11), with reports of human infections in Europe, Asia, Africa and the Middle East11. Imipenem MIC50/MIC90 values for the 206 ST164 isolates were 128/128, and 32/64 mg/L for the 213 GC2 isolates, indicating that the appearance of ST164 was responsible for the overall increase in carbapenem resistance between studies.

GC2 and ST164 were isolated throughout the study period and across the ICU (Fig. 1B). On 37/364 (10.2%) sampling occasions, GC2 and ST164 were isolated from the same bed unit. Despite their near-even prevalence in the ICU environment and patients, GC2 strikingly accounted for 12/13 (92.3%) clinical isolates obtained over the study period, with the remaining sputum isolates belonging to ST164. In contrast to GC2 and ST164, carbapenem-sensitive (CSAB) STs appeared sporadically over the course of the study and were generally localised to a single room, patient or time point (Fig. S5).

The GC2 population shifted significantly between studies through the introduction of multiple discrete sub-clusters

Consistent with our first study, carbapenem-resistance in GC2 CRAB isolates was conferred by the carbapenemase gene blaOXA-23. To determine the genomic contexts of these blaOXA-23 genes, we examined 19 hybrid-assembled complete GC2 genomes and screened the collection of 213 GC2 draft genomes with signature sequences indicative of the Tn2006 or Tn2009 insertions identified in our first study10. This revealed that the chromosomal positions encountered in the first study accounted for blaOXA-23 carriage in all GC2 isolates collected here (Fig. 2A; Supplementary Data 1). Additionally, one hybrid-assembled genome (DETAB-E2110) contained two copies of Tn2006, with the second copy found in the 13,545 bp plasmid pDETAB10/Tn2006 (Fig. S6). pDETAB10/Tn2006 was generated by the insertion of Tn2006 into the backbone of the 8731 bp R2-T1 type plasmid pDETAB10, which was present in 75/547 (13.7%) GC2 isolates in our first study and 175/213 (82.2%) GC2 isolates here. Screening this collection with the signatures of this insertion revealed that pDETAB10/Tn2006 was not present in any other isolates. DETAB-E2110 had MIC values of 128 and >128 mg/L for imipenem and meropenem, which were the equal-highest observed in this collection.

Although the GC2 isolates collected here shared Tn2006/Tn2009 insertions with the GC2 clusters examined in our previous study, chromosomal segments that contain transposons can be exchanged by homologous recombination between distinct A. baumannii strains12, so we sought phylogenetic evidence to assess cluster persistence in this ICU. To determine whether the GC2 population observed in this study was derived from that present in 2019, we constructed a phylogeny from the GC2 genomes obtained here, along with representative genomes for each of the 17 GC2 clusters identified in our first study (Fig. 2A). The phylogeny featured three large clusters (labelled x, y and z in Fig. 2A) that included 49, 72 and 92 GC2 genomes from this study. Each of the large clusters was separated from the others by long branches in the phylogeny. Representatives of 16 GC2 clusters from the 2019 study were confined to the diverse central cluster (x in Fig. 2A), with a single representative present in one of the two more clonal clusters, cluster 24 (C24) (y in Fig. 2A). Within the diverse central cluster, representatives from the 2019 study were largely distinct from isolates collected here, with the exception of C16, which differed from isolate DETAB-P494 by just one core-gene SNP (cgSNP). Overall, the GC2 population was more diverse in 2021 than it was in 2019, which is consistent with frequent and ongoing introductions of distinct GC2 clusters to the ICU. Further supporting this, some 2021 GC2 clusters (C18–23) appeared briefly in the ICU, and were present for no more than three sampling weeks (Fig. 2B). These will be referred to as transient GC2 clusters.

To detect putative patient-associated introductions to the ICU over the course of this study, we identified instances where, apart from in week 1, the first isolates of transient GC2 clusters appeared in patient samples. We found six such instances, involving six different clusters (Fig. 2C). Within transient clusters, isolates differed by 0–4 cgSNPs. The numbers of patient samples within each transient cluster (C18–C23) were 1, 5, 1, 7, 1 and 2, respectively (Fig. 2C). C22 was represented by a single patient isolate, but isolates of the other five clusters were found in two or more different environments each. These included the bed units occupied by the patients that initially carried the clusters, and either bed units within the same rooms (C18, C19, C21), bed units in different rooms (C23), or a communal cleaning cart (C20) (Fig. 2C). C19 and C21 were carried by two patients each, and the second patient that each cluster was isolated from most parsimoniously acquired C19/C21 from their contaminated room environments. All six transient GC2 clusters that first appeared in patient samples disappeared from the ICU following the departure of the last patient that carried them (Fig. 2C).

The largest GC2 clusters, C24 and C25, were present in most (10/13 for C24) or all (13/13 for C25) study weeks (Fig. 2B). The largest cluster, C25, only contained genomes from this study, all of which differed from one another by 0–6 cgSNPs (Supplementary Data 2). Isolates in C24 differed from one another by 0-11 cgSNPs, and from the 2019 study representative C2 genome by 5–10 cgSNPs (Supplementary Data 2). As representatives of C24 and C25 were already present in the first week of this study (Fig. 2B), we did not capture their introductions to the ICU, so cannot determine whether they were introduced in single or multiple events. However, the presence of sub-clusters of C24 and C25, containing isolates that do not differ at the cgSNP level (0 cgSNPs) but were found in multiple bed units or rooms, is evidence for the ongoing transmission of C24 and C25 within the ICU.

ST164: A dominant emerging clone recently introduced but fully established in the ICU

ST164 was not detected in this ICU in our first study. Over the course of this study, we obtained 206 ST164 isolates from 26 different sample types from patients and the ICU environment. The three most common positive sample types were oral (17/206, 8.3%), nasogastric tube (12/206, 5.8%) and rectal (12/206, 5.8%) swabs for patients, and bedside table (25/206, 12.1%), ventilator (22/206, 10.7%) and bed rail (16/206, 7.8%) swabs for the environment.

It was, therefore, important to establish whether this lineage had been introduced recently or had persisted in the ICU for an extended period. To assess their structural diversity, eight ST164 isolates were hybrid-sequenced to generate complete genomes. All eight complete genomes consisted of a 3.9 Mbp chromosome and five plasmids, pDETEC17–pDETEC21, that ranged from 12,790 to 2309 bp (Fig. 3A). Screening the entire collection of 206 ST164 short-read assemblies revealed that the five plasmids were present in almost all ST164 isolates (pDETAB19 195/206; pDETAB17 201/206; others 206/206).

All ST164 genomes contained five acquired antibiotic resistance genes (ARGs). Four of these encoded beta-lactamases: blaNDM-1, blaOXA-23, blaCARB-16 and ampC (also called blaADC). In the complete ST164 genomes, all ARGs were located in the chromosome (Fig. 3A). The blaNDM-1 gene was in the complex transposon Tn692413 inserted downstream of glmS, along with the bleomycin resistance gene bleMBL and the aminoglycoside resistance gene aphA6. The remaining beta-lactamase genes were in four ISAba1 composite transposons: blaOXA-23 in two copies of Tn2006, ampC in Tn6168, and blaCARB-16 in a novel transposon named Tn7735 (Fig. 3A). Screening draft genomes with the junction sequences associated with all five ARG-containing insertions revealed that they were totally conserved across this ST164 population.

The near-total conservation of mobile genetic elements and chromosomal insertion sites suggested that the ST164 population in this ICU was highly clonal. To confirm this, we determined cgSNPs between all isolates in this collection and found that they ranged from 0 to 21. We did not find evidence for the introduction of ST164 clusters with patients over the course of this study, and we were aware that the same ST had been isolated from a sputum specimen collected in this ICU in January 202114, four months prior to this study. We therefore used the genome of that clinical isolate, DETAB-R21, as a reference for a time-dated phylogeny for the genomes generated here (Fig. 3B). The time-dated phylogeny was consistent with this ST164 population having diversified from an initial introduction to the ICU in mid-2020. The two deepest branches in this phylogeny had estimated dates of divergence from a common ancestor in April 2020, giving rise to clusters 164-A and 164-B (Fig. 3B). DETAB-R21 clustered with 164-B. Both 164-A and 164-B were present in the first and last weeks of this study, but were not present in every intervening week, with no 164-A isolates in week 11 and no 164-B in weeks 3 and 12 (Fig. 4). Clusters 164-C (present in weeks 5-13) and 164-D (weeks 2, 5-10), emerged from 164-A and 164-B, respectively (Fig. 3B), and were not present in the ICU at the outset of the study (Fig. 4). Notably, the first isolates of 164-C and 164-D were derived from environmental samples rather than patient samples, consistent with their emergence from a pre-existing ICU population rather than from patient-associated introductions over the course of this study.

Over the course of the study, we observed 10 clear instances where patients were admitted to the ICU, were A. baumannii-negative at one or more sampling points, but then yielded ST164 from subsequent oral, rectal or tube swabs. All of these patient-derived ST164 isolates were identical (0 cgSNPs; Supplementary Data 3) to one or more isolates that had previously been obtained from the ICU environment or from other ICU patients. We conclude that these events represent acquisition of ST164 from the population persisting in the ICU. Acquisition events involved three of the four ST164 phylogenetic clusters: ST164-A in 3/10 cases, ST164-B in 4/10, and ST164-C in 3/10. In one further case, a patient who produced GC2 from their first screening samples appeared to acquire ST164-D, which was isolated from a subsequent oral swab and a clinical sputum sample.

Persistence of CRAB in the ICU results in clinically relevant CRAB acquisition by patients

Using the phylogenetic evidence described above, we sought to determine the derivation of each of the 13 CRAB clinical isolates collected over the course of this study and, therefore, to identify the routes through which they might have been acquired by these 10 patients over the study period. Most (12/13) of the clinical isolates were GC2, and amongst these, 10 were C25, one was C24, and one was C21. The remaining clinical isolate was ST164-D. Representatives of all four of these clusters were present in multiple patient and environmental samples prior to their appearance in clinical samples, but to avoid any ambiguity associated with small numbers of cgSNPs (≤4), we focused on comparing identical (0 cgSNP) isolates when describing clinically-relevant CRAB acquisition in the ICU.

The GC2 C21 cluster appears to have been introduced to the ICU by patient 200 (P200) (see above), who was admitted to the ICU from either the community or a different hospital (data not available). The presence of C21 in a P200 clinical sample is, therefore, indicative of acquisition outside this hospital, before the development of clinically relevant symptoms requiring sputum sample collection in this ICU. The GC2 C24 isolates obtained from P172 were identical to isolates obtained from P172’s first screening samples on arrival to the ICU, which might suggest that they carried C24 on arrival. However, because C24 was circulating in the ICU (see above), and identical isolates had been obtained from other patients and the ICU environment prior to the admission of P172, it is also possible that P172 acquired C24 in the ICU before their first screening samples were taken.

In the remaining eight cases, patients had been CRAB-negative at one or more sampling points prior to the development of symptoms that necessitated clinical sample collection. In three of these cases, isolates identical to the clinical isolates (0 cgSNPs) had previously been isolated from the ICU environment. It, therefore, seems most parsimonious that these patients acquired CRAB in the ICU. The five remaining clinical isolates were not identical to any other isolates in the collection but differed from multiple isolates by ≤4 cgSNPs. We conclude that acquisition in the ICU was responsible for the majority (9 or 10/10) of CRAB found in clinically relevant patient samples over the course of this study.

Global epidemiology and carbapenemase gene distribution in ST164

To explore the global distribution of ST164 isolates, we chose four representative strains from our study to represent the total diversity observed in this ICU and further incorporated 131 ST164 publicly-available genomes that were collected in 26 countries from five continents between 2017 and 2023 (Supplementary Data 4). Overall, ST164 strains are primarily distributed in Asian countries (Fig. 5C). Most publicly-available ST164 genomes (95.6%; 129/135) were isolated from patients, and the highest numbers of isolates were collected between 2016 and 2019 (Fig. 5A and B). The most common KL type in global ST164 isolates was KL47 (63.4%, 83/131), which differs from the GC2 isolates where KL30 (43.2%, 92/213) and KL93 (33.8%, 72/213) were most common. ST164 genomes could be further divided into ST234 (20%, 27/135), ST1418 (54.8%, 74/135) and novel types (25.2%, 34/135) using the Oxford MLST scheme (Fig. 5A). ST164 genomes differed by between 3 and 6,037 cgSNPs (Supplementary Data 5), and could be divided into seven main clades via evolutionary branches (Fig. 5A). All isolates assigned to Clade 1 were from the USA and carried the blaNDM-1 cabapenemase gene. Isolates from Clade 6 exhibited the widest geographical distribution, with representatives isolated on five different continents. The largest cluster, Clade 7, is dominated by strains isolated from Thailand. Notably, strains collected in China formed a monophyletic cluster, Clade 2. Importantly, Clade 2 strains harboured the highest number of carbapenemase genes, which were carried in multiple transposons, including Tn6924, Tn7735, and up to three copies of Tn2006. Isolates of other clades (clade 1, clade 3–7) carry at most one carbapenem resistance gene. Moreover, all strains from clade 1 carry blaNDM-1, and the majority of clade 3–7 isolates only harboured blaOXA-23. Co-carriage of blaNDM-1 and blaOXA-23 has so far only been seen in China and Malaysia.