Multilocus sequence typing of clinical and colonizing isolates of Acinetobacter baumannii and comparison with the world isolates

Abstract

Objectives

To establish the epidemiological correlation between the study isolates, Indian and World isolates of Acinetobacter baumannii by performing Multilocus Sequence typing.

Materials and methods

A total of 181 isolates (Sputum (n = 116), lower respiratory tract other than sputum (n = 36), upper respiratory tract (n = 20), Environment (medical) (n = 4), and Blood (n = 5) of Acinetobacter baumannii were retrieved from our repository. DNA was isolated and Multilocus Sequence Typing was performed according to the Pasteur scheme. The amplified fragments were sequenced by outsourcing, and the locus and the sequence types were determined as given in the PUBMLST site. The clonal complexes were assigned using eBURST.

Results and conclusion

Of the 181 isolates, 20 were colonizers and 4 were from hospital environments. All the study isolates were multidrug-resistant (MDR) and 4 of them were extensively drug-resistant (XDR). 23 sequence types were unique and were assigned new sequence types. Among them, 2125 (n = 12), an SLV (Single Locus Variant) of 2, was the commonest followed by 2126 (n = 2) which was a DLV (Double Locus Variant) of 2 and an SLV of 2125. Others were singletons. Among the known STs, 149 (n = 72) was the commonest followed by ST 2 (n = 62) & 415 (n = 5), ST 10 (n = 4), ST 15, ST622 and ST1482 (3 each). ST149 had 1SLV ST1482 (n = 3). ST 2 has 5 SLVs (415, 1555, 2125, 2128, & 2131, and 2 DLVs (2130 & 2126). eBURST analysis of the study isolates showed three groups: Group I (86 isolates) with ST 2 as the primary founder, group II (6 isolates) and group III (79 isolates) with ST 149 as the primary founder. All the other 11 isolates were singletons. There was no difference in antimicrobial sensitivity or sequence types of clinical and colonizing isolates. The sequence types of study isolates were compared to the world isolates in the PUBMLST database. To conclude, MLST is an important tool for establishing isolates’ phylogenetic relationships. Acinetobacter baumannii being an important nosocomial pathogen should be routinely screened for the frequent changes in the sequence types to demonstrate the emerging resistance patterns and other changes.

Introduction

Acinetobacter baumannii (A. baumannii) is an aerobic gram-negative coccobacillus. A. baumannii belongs to “ESKAPE” six pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) which are known for their multidrug resistance and virulence. This group is responsible for most nosocomial infections and can escape the biocidal effect of antimicrobial agents [1]. A. baumannii is an important nosocomial pathogen responsible for a wide range of human infections. It accounts for ~ 2% of healthcare-associated infections in the Western world and this number is double in Asia and the Middle East [2]. Irrational use of antibiotics and selective pressure has led A. baumannii to emerge as an antibiotic-resistant pathogen in both hospital and environmental settings [3]. In some immunocompromised patients, it can also be found as a colonizer [4].

Various genotypic methods are used for Acinetobacter strain typing, like plasmid profile, DNA restriction fragment length polymorphisms determined by pulsed-field gel electrophoresis (PFGE), Repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR), and Multilocus sequence typing (MLST). [5]. Average nucleotide identity (ANI) for identification along with MLST based on Next Generation Sequencing (NGS) is the most promising method. The whole Genome sequencing (WGS) for taxonomic classification is rapid and accurate. These typing techniques are now being used to show a relationship between the nosocomial spread of infection and the source [6]. Multilocus sequence typing (MLST) is a molecular typing method used to derive phylogenetic relationships among A. baumannii strains for epidemiological investigations. Pasteur and Oxford are the two types of MLST schemes used for genomic sequencing [7]. Seven housekeeping genes; gltA, fusA, pyrG, recA, cpn60, rplB and rpoB are studied under the Pasteur scheme [8]. ST 2 is the most common sequence type existing worldwide and is reportedly multidrug resistant. Hence, the objective was to study the frequency of different sequence types of A. baumannii in clinical and surveillance isolates. And to compare it with the Indian and World isolates concerning the site of isolation to show epidemiological correlation.

Methods

Clinical isolates

This is a retrospective analytical study including a total of 181 isolates of Acinetobacter baumannii present in the repository (Which is a collection of all the clinical and environmental isolates identified in the laboratory, preserved in 16% glycerol broth at -80 °C), isolated during 2018–2021. These isolates were from clinical samples of patients (including sputum, bronchial aspirate, bronchioalveolar lavage, endotracheal aspirate, blood, and pleural fluid), and surveillance samples (nasal and throat swabs) taken from patients and healthcare workers. Isolates from environmental samples taken from hospital beds, ventilators, and bed railings were also included. All the isolates were collected from ICU and the wards of our hospital which is a respiratory diseases institute.

Bacterial isolates

The isolates were revived on blood agar plates and reconfirmed as A. baumannii using MALDI-TOF MS(Bruker Daltonics, Bremen, Germany) and/or blaOXA-51 PCR. All the isolates were subjected to antimicrobial susceptibility testing according to CLSI 2020 guidelines by Kirby Bauer disc diffusion method and/or Vitek 2 for Ampicillin-sulbactam, Piperacillin, Piperacillin-tazobactam, Ceftazidime, Cefepime, Cefotaxime, Meropenem and Imipenem. Colistin MIC was tested by the micro broth dilution method. All the isolates included in the study were MDR (resistant to more than three different classes of antimicrobials) or XDR (Extensively drug resistant-MDR plus resistant to Colistin). The research was cleared up by the Institutional Human Ethics Committee.

Multilocus sequence typing

Multilocus Sequence Typing was performed according to the Pasteur scheme. Fragments of seven housekeeping genes cpn60, fusA, gltA, pyrG, recA, rplB and rpoB were amplified using the primers and conditions for PCR as given in the site https://pubmlst.org/organisms/ Acinetobacter-baumannii) with a few changes in annealing temperature of fusA, recA and ropB, (Table 1). The amplified fragments were sequenced by outsourcing (Eurofins Genomics India Pvt Ltd., Bangalore-560048). The final volume of 50 µl of amplification mixture had 1.25U Taq polymerase, 1X of PCR buffer (with MgCl₂), 10pM each of primers, 1.25mM of dNTPs and 2 µl of DNA template. Allele sequences were compared to those given in the database (http://pubmlst.org/abaumannii/) to determine the alleles and sequence types (ST).

Table 1 Primers for MLST and PCR conditions used

Using eBURST (http://pubmlst.org/analysis) clonal complexes (CCs) were assigned. They were further defined as single-locus variants (SLVs) and double-locus variants (DLVs). All the A.baumannii isolates in the PUBMLST site tested by the Pasteur scheme were further analyzed according to the country, isolate type etc.

Results

Study isolates

MLST was performed in 181 isolates using the Pasteur scheme. The isolates were from Sputum (N = 116), lower respiratory tract other than sputum (N = 36), upper respiratory tract (N = 20), Environment-medical (N = 4), and Blood (N = 5). All our isolates except 4 (XDR) were multi-drug resistant including the colonising and hospital environment isolates. Of the 181 isolates, 23 were assigned new sequence types. Among them, 2125 (n = 12), an SLV of 2, was the commonest followed by 2126 (n = 2) which was a DLV of 2 and an SLV of 2125. Others were singletons (Table 2). Among the known STs, 149 (n = 72) was the commonest followed by ST 2 (n = 62), and ST149 had 1SLV ST1482 (N = 3). ST 2 has 5 SLVs (415, 1555, 2125, 2128, & 2131, and 2 DLVs (2130 & 2126) (Table 2). All ST 2 isolated in this study were found to be positive for blaOXA-23, the presence of which is a mechanism of multidrug resistance in Acinetobacter baumannii.

Table 2 Sequence types of Acinetobacter baumannii isolates from this study (n = 181)

eBURST analysis was performed on all the A.baumannii isolates in the PUBMLST database which were tested by the Pasteur scheme.

eBURST analysis of the study isolates showed three groups: Group I (86 isolates) with ST 2 as the primary founder, group II (6 isolates) and group III (79 isolates) with ST 149 as the primary founder. All the other 11 isolates were singletons (Fig. 1 in supplementary file). Similar observations were made in the minimum spanning tree deduction of the isolates (Fig. 1).

Fig. 1

Minimal spanning tree (MS Tree) of MLST data of Study Acinetobacter baumannii isolates

Indian isolates

There were 304 isolates (accessed on 25th October 2023) of A. baumannii in the PUBMLST site of which 275 isolates of the STs (Pasteur scheme) are known. They are from respiratory isolates (sputum-127, LRT(Lower respiratory tract)-40, Blood (43), upper respiratory tract (34), Wound (8), urine (2), CSF (Cerebrospinal fluid) (1), environment-Medical (6), Environment (3) and others (23), unknown (21). Burst analysis showed that 224 out of 265 isolates belonged to 7 groups and the rest 33 were singletons. Group II was the largest with 107 isolates with ST 2 as the founder. The second largest was group 5 with 88 isolates and ST149 with 73 isolates was the primary founder with 3 SLVs (ST1482, ST2480, and ST622) and one DLV (ST2129). 168 isolates were from the lower respiratory tract including sputum. Twenty-six isolates were from blood. 14 of these were from Kolkata, India. ST 2 was the commonest with 9 isolates. Interestingly none of the blood isolates belonged to ST 149. Most Indian isolates belonged to ST2 followed by ST 149. A minimal spanning tree (MS Tree) of MLST data showing Indian Acinetobacter baumannii isolates is given in Fig. 2.

Fig. 2

Minimal spanning tree (MS Tree) of MLST data on Indian isolates of Acinetobacter baumannii depicting the source and STs

World isolates

PUBMLST website had 7379 A. baumannii isolates (accessed on 25th October 2023) out of which STs of 5383 were by the Pasteur scheme. The isolates were from various samples viz., Lower respiratory tract including sputum (1153), Blood (800), IV catheters (85), Environment (Medical) (114), Environment (451), Stool/rectal swab (234). Urine (311), CSF (65) and Wound (8). Asia was the largest contributor with 2374 isolates followed by Europe (1165) and North America (1041). Most of the isolates were from the USA (913) followed by China (817), Brazil (418), Germany (323), Russia (285) and India (263) etc. ST 2 was the commonest with 1466 isolates, followed by ST1 (213), ST 25 (110), ST79 (101), ST15 (99), ST149 (79) and ST 415 (18). Since the isolates submitted to the database by countries are disproportionate, the results may be skewed. However, it gives a fair idea that most STs are prevalent around the globe and are from varied samples. A comparison of the ST2 and ST149 of the world, Indian and study isolates showed that ST2 was the most common among the Indian and World isolates. However, ST 149 was the most common in the study isolates (Fig. 3). A minimal spanning tree (MS Tree) of MLST data showing a population snapshot of the world Acinetobacter baumannii isolates is given in Fig. 4.

Fig. 3

Percentage distribution of ST 2 and ST 149 in Study, Indian and World isolates

Fig. 4

Minimal spanning tree (MS Tree) of MLST data showing population snapshot of the world Acinetobacter baumannii isolates

Lower respiratory tract including sputum

Out of 1152 isolates, the isolates were predominantly from Asia (840) followed by N. America (181), Europe (64), S. America (28) Africa (26) and Oceania (13). China (375), USA (173) and India contributed the most isolates. Here again, ST 2 was the commonest (396), followed by ST 149 (53) and ST1 (25).

Blood

There were 800 isolates from Blood distributed in Asia (387), S. America (191), and Europe (84). Among the countries, Brazil (171) ranked the highest followed by China (113), and Turkey (97). There were 28 isolates from India. ST2 was the most common followed by ST1 and ST79.

IV catheters

Eighty-five isolates from the IV catheter were from S. America (27) followed by Asia (16), Europe (11), N. America (11) and Africa (2). Brazil (17), China (10), and Mexico (10) had the most. Burst analysis showed 6 groups and 29 singletons. Out of these Group 3 was the largest with 14 isolates and 9 ST, 25 was the founder having 3 SLVs (300, 402 & 991) and 2 DLVs (113 & 1625). There were no Indian Isolates. ST2 (23), ST1 (5), ST15 (4), and 25 (3) were predominant.

CSF

There were 65 isolates from CSF with known STs belonging to 7 groups and 6 singletons. Group 2 was the largest with 20 isolates. ST2 was the founder member of this group. Most of the isolates were from Brazil (32), followed by China (10) and Bangladesh (4). ST 2 (18) was the most abundant followed by ST 15 & 79.

Medical environment

The majority of the 114 isolated from the medical environment were from Asia(61) followed by Europe (34) & Spain (29). There were 8 groups and 49 singletons. Group 1 was the largest with 24 isolates with 6 STs and ST2 as its founder with 5 SLVs (254, 257, 261, 263 & 600). The isolates were mainly from Spain (29), Jordan (24), China (20), Vietnam (9), USA (8) and India (6).

Environment

Four hundred and fifty-one isolates were from the environment, namely chicken farm stock, soil, water, plant root, and shopping cart. The isolates were predominantly from Europe (282) followed by Asia (54). The majority were from Norway followed by Germany, Nigeria, and Jordan. All the chicken farm stock samples were from Norway. Four hundred and fifty-one isolates belonging to 19 groups were from the environment. Group 3, with 207 isolates, was the largest with ST562 as the founder and was most diverse with 11 STs. ST1369, ST1371, ST 1354, and ST 2 (10) were prominent.

Stool / rectal swabs

The database had 234 isolates from stool / rectal swabs. The majority (131) were from North America. Among the countries, the USA (129), South Korea (24), Brazil (20), France (22), and China (4) were dominant. 13 groups were identified and 44 were singletons. Group 2 was the largest with 65 isolates. ST 416 was the founder. This was followed by group 2 with ST2 as the founder.

Urine

The database had 250 urinary isolates predominantly from Asia, especially China. The isolates belong to 16 groups and as many singletons. Group 2 was the largest with ST33 (120 isolates) as the founder. ST 2 was the next commonest with 54 isolates and predominantly from Asia. There were only 2 urinary isolates from India.

Discussion

Molecular typing is an important tool to determine the genetic and epidemiological relatedness of A. baumannii.

In the present study, 181 isolates were typed by MLST (Pasteur Scheme). ST149 was the most common followed by ST2 and its SLVs. However, ST 2 belonging to international clone 2 was the dominant sequence type in the world, which is comparable to the Indian and world scenario. There were no STs from the international clone 1 or 3. ST2 with 6803 isolates (as of 25th Jan 2024) is distributed in all the continents of the world with predominance in N. America followed by Asia. Among the countries, the USA ranked first with 3355 followed by China (1339), Australia (240), and India (220) among others. These isolates were majorly from Sputum followed by blood (189). Five SLVs of ST2 are ST415, 1555, 2125, 2128, and 2131. Twenty-one isolates of ST 415 were encountered from N. America (16) and Asia (5). The first isolate appeared in 2008. However, ST 1555 with 10 isolates was seen only in Asia especially in China (9) and India (1). The other 3 SLVs were restricted to our hospital. They have not disseminated since.

ST149 with 120 isolates was predominant in the present study (72) and has been isolated from Nepal (38), other parts of India (7), and one each from Norway, Japan and the UK. Seventy of our isolates were from the respiratory tract. This originated in 2005 in Japan and was later seen in Nepal in 2013-14. In the present study, isolates were seen from 2015 onwards. Due to frequent travel between the two countries, it is possible that it spread later to India. Similarly, the SLV of ST 1482 having 3 isolates was from our study Table 2. This genetic variation occurred locally. Being a respiratory disease hospital, people from all parts of the country and neighbouring countries visit it and hence dissemination.

Out of the 23 new STs found in the present study, STs 2125, 2126 2128 and 2131 were all either SLVs or DLVs of ST2. Hence, ST 2 has undergone genetic variation to produce new types that are related to it. Except for ST415 and ST1555, none of the others have been found elsewhere. ST 415 with 21 isolates was predominantly isolated from the USA (16) in 2006-7 followed by India (5) (all from the present study) in 2015 and were from the respiratory tract and were XDRs.

The next common STs after ST2 in the world were ST 1 (605) followed by ST 437 (532), ST499 (272), ST25 (225), ST79, ST 3, and ST 10.

Among these international clones 2, 149, 415, 1482, 94 and 1555 were found in the present study.

Among these ST1550 was the most abundant with 35 isolates followed by ST415(18), ST605 (15), ST 2125 (12) and ST1150 (10) However, new ST 2125, an SLV of ST2 found in the present study with 12 isolates was unique. The isolates were from sputum (7), upper respiratory tract (4) and Blood (1).

ST 415 is a SLV of ST2 with 21 isolates and 14 SLVs. ST 2125 is the largest SLV (12 isolates) of ST 415 and all were from India. They were isolated from sputum (7), Upper respiratory tract (4) and Blood (1). ST 415 is the SLV of ST2 where the allele recA − 2 has been replaced with 248.

The presence of 2690 STs, from across the world, in the PUBMLST database shows that A. baumannii is evolving and giving rise to new sequence types. However, the majority.

are still a few clones.

Most of our isolates were multi-drug resistant with varied sequence types. Published data from Thailand and neighbouring countries showed that ST2 of the clonal complex (CC) 2 was most abundant and multidrug-resistant. [9, 10].

CC2 of the Pasteur scheme corresponds to CC92 in the Oxford scheme and international clone 2. This was the most widely distributed CC of carbapenem-resistant isolates with bla_{OXA−23−like} producing CC92, which incorporates ST136 and its several single-locus variants. Interestingly, isolates belonging to CC92 were more resistant compared to other STs. Overall, these observations suggest a wide distribution of carbapenem-resistant and blaOXA-23-like producing clone CC92, especially ST92, ST75 and ST138, as the principal reason for the rapidly increasing carbapenem resistance rate in China [11]. All our ST2 isolates produced bla_{OXA−23−like} and could explain that they were all MDR.

Extrapolating this observation, it can be inferred that ST2 of international clone 2 would be multidrug, especially Carbapenem-resistant. This was seen in the present study also.

Conclusions

To conclude, multi-drug-resistant clones of A. baumannii belonging to various STs are circulating worldwide. It should be limited by implementing antibiotic and infection control policies in every hospital to reduce transmission through healthcare workers and medical devices.