A total of 401 confirmed Gram-negative MDR-ESKAPEE and 30 confirmed Gram-positive MDR-ESKAPEE pathogens were identified from Queen Sirikit Naval Hospital from 2017 to 2018 (Table
1). These were confirmed, non-duplicated MDR isolates that have not been published.
E. coli (41.3%) was the most common pathogen identified, followed by
K. pneumoniae (23.7%) and
A. baumannii (22.5%). Antibiotic resistant phenotypes of 401 Gram-negative MDR-ESKAPEE were as follows; carbapenem resistant isolates 204/401 (51%), ESBL-only producers 182/401 (45%), ESBL producers and colistin resistant 7/401 (2%), and non-ESBL producers 8/401 (2%). Among the 204 carbapenem resistant isolates, a high percentage of
A. baumannii (47.5%) and
K. pneumoniae (33.4%) were identified, while the majority of
E. coli were ESBL-only producers (82.4%). There were 6
E. coli and 1
K. pneumoniae that were both ESBL producers and colistin resistant due to the carriage of
mcr genes
. Eight MDR isolates (6
E. coli and 2
K. pneumoniae) that were resistant to other antibiotic classes were also identified (Table
1). Of 30 Gram-positive MDR-ESKAPEE isolates, all
E. faecium 5/5 (100%) were VRE and 21/25 (84%)
S. aureus were MRSA.
Table 1
Distribution of pathogens and antimicrobial resistance phenotypes of 431 ESKAPEE isolates collected in Chonburi, Thailand in 2017–2018
E. faecium | 5 (1.2%) | N/A | N/A | N/A | N/A | 5 (100%) | N/A |
S. aureus | 25 (5.8%) | N/A | N/A | N/A | N/A | N/A | 21 (100%) |
K. pneumoniae | 102 (23.7%) | 68 (33.4%) | 31 (17%) | 1 (14.3%) | 2 (25%) | N/A | N/A |
A. baumannii | 97 (22.5%) | 97 (47.5%) | 0 | 0 | 0 | N/A | N/A |
P. aeruginosa | 20 (4.6%) | 19 (9.3%) | 1 (0.6%) | 0 | 0 | N/A | N/A |
Enterobacter spp. | 4 (0.9%) | 4 (2%) | 0 | 0 | 0 | N/A | N/A |
E. coli | 178 (41.3%) | 16 (7.8%) | 150 (82.4%) | 6 (85.7%) | 6 (75%) | N/A | N/A |
Total No. (%) | 431 (100%) | 204 (100%) | 182 (100%) | 7 (100%) | 8 (100%) | 5 (100%) | 21 (100%) |
Antibiotic resistance phenotypes
A variety of antibiotic resistance phenotypes were identified among the collected MDR ESKAPEE pathogens. Seventy to one hundred percent of the Gram-negative isolates were resistant to β-lactam antibiotic classes (cephems, monobactams and β-lactam inhibitors), a summary of antibiotic resistant phenotypes is provided in Table
2. MDR-
E. coli isolates were more sensitive to amikacin (96%) compared to tobramycin (59%) and gentamicin (42%), however, all
K. pneumonia and
P. aeruginosa were sensitive to amikacin. Six
E. coli and 1
K. pneumoniae were resistant to colistin. Of these, 5
E. coli isolates were isolated from urine, 1 from pus, and
K. pneumoniae was from sputum (Additional file
2: Table S2). However, the resistance to polymyxin was relatively low indicating that the resistance was not as widespread in this area. More than 94% of
A. baumannii were resistant to five antibiotics classes (carbapenems, cephems, monobactams, β-lactam inhibitors, and 5-fluoroquinilones; Table
2). Nineteen out of 21 MRSA isolates were resistant to ciprofloxacin and 12 were resistant to tetracycline. Of note, all 21 MRSA isolates (100%) were resistant to clindamycin, 1 isolate was resistant to mupirocin and 4 isolates were resistant to Trimethoprim-Sulfamethoxazole (Table
3). All 5 VRE isolates were resistant to the 14 antibiotics tested, including the first-line treatment for VRE: ampicillin, gentamycin, and linezolid (Table
3). One VRE was resistant to linezolid but susceptible to quinupristin-dalfopristin and nitrofurantoin while another isolate was resistant to quinupristin-dalfopristin but susceptible to linezolid. No VISA or VRSA isolates were identified in this study.
Table 2
Resistance to tested antibiotics in Gram-negative ESKAPEE bacterial isolates
Carbapenem | Ertapenem | 196 (49) | 55 (54) | 97 (100) | 20 (100) | 4 (100) | 20 (11) |
Imipenem | 178 (44) | 58 (57) | 95 (98) | 14 (70) | 0 | 11 (6) |
Meropenem | 176 (44) | 59 (58) | 95 (98) | 12 (60) | 0 | 9 (5) |
Cephem | Cefazolin1 | 401 (100) | 102 (100) | 97 (100) | 20 (100) | 4 (100) | 178 (100) |
Cephalothin1 | 395 (98) | 99 (97) | 97 (100) | 20 (100) | 4 (100) | 178 (100) |
Cefuroxime2 | 386 (96) | 99 (97) | 97 (100) | 20 (100) | 4 (100) | 166 (93) |
Cefoxitin2 | 344 (86) | 59 (58) | 97 (100) | 20 (100) | 4 (100) | 164 (92) |
Cefotaxime3 | 398 (99) | 102 (100) | 94 (97) | 20 (100) | 2 (50) | 178 (100) |
Ceftazidime3 | 378 (94) | 99 (97) | 94 (97) | 15 (75) | 3 (75) | 167 (94) |
Ceftriaxone3 | 379 (95) | 97 (95) | 94 (97) | 20 (100) | 4 (100) | 164 (92) |
Cefepime4 | 373 (93) | 99 (97) | 94 (97) | 15 (75) | 3 (75) | 162 (91) |
Monobactam | Aztreonam | 380 (95) | 99 (97) | 97 (100) | 14 (70) | 3 (75) | 167 (94) |
β-Lactam inhibitor | Ampicillin | 397 (99) | 102 (100) | 97 (100) | 20 (100) | 4 (100) | 174 (98) |
Ampicillin-Sulbactam | 401 (100) | 102 (100) | 97 (100) | 20 (100) | 4 (100) | 178 (100) |
Amoxicillin-Clavulanate | 388 (97) | 100 (98) | 97 (100) | 20 (100) | 4 (100) | 167 (94) |
Piperacillin | 389 (97) | 102 (100) | 97 (100) | 10 (50) | 2 (50) | 178 (100) |
Piperacillin-Tazobactam | 188 (47) | 71 (70) | 94 (97) | 6 (30) | 3 (75) | 14 (8) |
5-Fluoroquinolone | Ciprofloxacin | 348 (87) | 86 (84) | 94 (97) | 14 (70) | 3 (75) | 151 (85) |
Levofloxacin | 341 (85) | 88 (86) | 91 (94) | 10 (50) | 2 (50) | 150 (84) |
Moxifloxacin | 355 (88) | 86 (84) | 97 (100) | 20 (100) | 2 (50) | 150 (84) |
Norfloxacin | 349 (87) | 84 (82) | 94 (97) | 14 (70) | 4 (100) | 153 (86) |
Folate Antagonist | Trimethoprim | 326 (82) | 84 (82) | 97 (100) | 20 (100) | 0 | 125 (70) |
Trimethoprim-Sulfamethoxazole | 300 (75) | 87 (85) | 74 (76) | 20 (100) | 0 | 119 (67) |
Aminoglycoside | Gentamicin | 249 (62) | 44 (43) | 88 (91) | 11 (55) | 3 (75) | 103 (58) |
Amikacin | 94 (23) | 0 | 85 (88) | 0 | 2 (50) | 7 (4) |
Tobramycin | 228 (57) | 73 (72) | 69 (71) | 11 (55) | 2 (50) | 73 (41) |
Tetracycline | Tetracycline | 341 (85) | 94 (92) | 88 (91) | 20 (100) | 2 (50) | 137 (77) |
Nitrofurantoin | Nitrofurantoin | 201 (47) | 61 (60) | 97 (100) | 20 (100) | 4 (100) | 7 (4) |
Chloramphenicol | Chloramphenicol | 196 (49) | 26 (25) | 97 (100) | 20 (100) | 0 | 53 (30) |
Polymyxin | Colistin | 7 (1.7) | 1(1) | 0 | 0 | 0 | 6 (3) |
Table 3
Resistance to tested antibiotics in Gram-positive ESKAPEE bacteria isolates
Aminoglycoside | Amikacin | 5 (17) | 5 (100) | 0 (0) |
Gentamicin | 14 (47) | 5 (100) | 14 (56) |
Tobramycin | 19 (63) | 5 (100) | 14 (56) |
β-Lactam inhibitor | Amoxicillin-Clavulanate | 21 (70) | 0 (0) | 21 (84) |
Ampicillin | 30 (100) | 5 (100) | 25 (100) |
Oxacillin | 21 (70) | 0 (0) | 21 (84) |
Natural penicillins | Penicillin G | 25 (83) | 0 (0) | 25 (100) |
Cephem | Cefoxitin | 5 (17) | 5 (100) | 0 (0) |
5-Fluoroquinolone | Ciprofloxacin | 24 (80) | 5 (100) | 19 (76) |
Lincosamide | Clindamycin | 26 (87) | 5 (100) | 21 (84) |
Macrolide | Erythromycin | 26 (87) | 5 (100) | 21 (84) |
Fusidane | Fusidic acid | 5 (17) | 5 (100) | 0 (0) |
Oxazolidinone | Linezolid | 1 (3) | 1 (20) | 0 (0) |
Antibacterials, Topical | Mupirocin | 1 (3) | 0 (0) | 1 (4) |
Mupirocin High level | 1 (3) | 0 (0) | 1 (4) |
Nitrofurantoin | Nitrofurantoin | 4 (13) | 4 (80) | 0 (0) |
Streptogramin | Quinupristin-dalfopristin | 1 (3) | 1 (20) | 0 (0) |
Ansamycin | Rifampin | 2 (7) | 0 (0) | 2 (8) |
Tetracycline | Tetracycline | 17 (57) | 5 (100) | 12 (48) |
Folate Antagonist | Trimethoprim | 10 (33) | 5 (100) | 5 (20) |
Trimethoprim-Sulfamethoxazole | 9 (30) | 5 (100) | 4 (16) |
Glycopeptide | Vancomycin | 5 (17) | 5 (100) | 0 (0) |
Teicoplanin | 5 (17) | 5 (100) | 0 (0) |
Antibiotic resistance genotypes
A wide variety of antibiotic resistance genes were identified from the isolates collected in this study, including all five major carbapenemase resistance genes (
blaKPC,
blaNDM,
blaOXA-48-like,
blaIMP and
blaVIM) and two colistin resistance genes (
mcr-1 and
mcr-3). The majority of
A. baumannii isolates harbored
blaOXA-23 (94/97, 97%), which represents 23% of all Gram-negative isolates collected.
K. pneumoniae,
E. coli, P. aeruginosa and
Enterobacter spp. isolates were shown to harbor
blaNDM (69/401, 17%) and
blaOXA-48-like (51/401, 13%) carbapenemase genes, with
K. pneumoniae carrying the highest percentage (Table
4).
K. pneumoniae and
P. aeruginosa isolates were shown to harbor
blaIMP (1/201, 0.5%) and
blaVIM (2/20, 10%), respectively. ESBL-producing isolates harbored a variety of ESBL genes with
blaCTX-M group 1 (182/401, 45%) being the most common in
K. pneumoniae, A. baumannii, and
E. coli (Table
4).
Table 4
Antibiotic resistance genes in ESKAPEE bacteria isolates
Carbapenemase genes | NDM | N/A | N/A | 56 (55%) | 4 (4%) | 1 (5%) | 1 (25%) | 7 (4%) | 69 (17%) |
KPC | N/A | N/A | 0 | 0 | 0 | 0 | 1 (0.5%) | 1 (0.25%) |
OXA-48 like | N/A | N/A | 48 (47%) | 0 | 0 | 0 | 2 (1%) | 50 (13%) |
IMP | N/A | N/A | 1 (1%) | 0 | 0 | 0 | 0 | 1 (0.25%) |
VIM | N/A | N/A | 0 | 0 | 2 (10%) | 0 | 0 | 2 (0.5%) |
OXA-23 | N/A | N/A | 0 | 93 (97%) | 0 | 0 | 0 | 93 (23%) |
OXA-24 | N/A | N/A | 0 | 0 | 0 | 0 | 0 | 0 |
β-lactamase genes | TEM | N/A | N/A | 84 (82%) | 77 (79%) | 0 | 3 (75%) | 63 (35%) | 227 (57%) |
SHV | N/A | N/A | 31 (30%) | 0 | 0 | 0 | 0 | 31 (8%) |
OXA | N/A | N/A | 18 (18%) | 0 | 0 | 3 (75%) | 42 (24%) | 63 (16%) |
CTX-M group 1 | N/A | N/A | 91 (89%) | 2 | 0 | 3 (75%) | 86 (48%) | 182 (45%) |
CTX-M group 9 | N/A | N/A | 4 (4%) | 0 | 0 | 0 | 68 (38%) | 72 (18%) |
CTX-M group 8/25 | N/A | N/A | 1 (1%) | 0 | 0 | 0 | 0 | 1 (0.25%) |
DHA | N/A | N/A | 4 (4%) | 0 | 0 | 0 | 4 (2%) | 8 (2%) |
CMY | N/A | N/A | 14 (14%) | 0 | 0 | 0 | 13 (7%) | 27 (7%) |
PER | N/A | N/A | 0 | 3 (3%) | 1 (5%) | 0 | 0 | 4 (1%) |
VEB | N/A | N/A | 0 | 2 (2%) | 5 (25%) | 0 | 1 (0.5%) | 8 (2%) |
Colistin resistance genes | mcr-1 | N/A | N/A | 1 (1%) | 0 | 0 | 0 | 3 (1.6%) | 4 (1%) |
mcr-3 | N/A | N/A | 0 | 0 | 0 | 0 | 1 (0.5%) | 1 (0.25%) |
mcr-1, mcr-3 | N/A | N/A | 0 | 0 | 0 | 0 | 2 (1%) | 2 (0.5%) |
Methicillin resistance gene | mecA | N/A | 21 (84%) | N/A | N/A | N/A | N/A | N/A | 21 (70%) |
Vancomycin resistance gene | vanA | 5 (100%) | N/A | N/A | N/A | N/A | N/A | N/A | 5 (17%) |
All 431 short-read WGS data and 2 long-read WGS data of plasmid in
E. coli with
mcr genes were submitted to NCBI database and is available as BioProject: PRJNA814829. The MLST data of WGS, BioSample accession number, and other information about bacterial isolates are reported in Additional file
3: Table S3. The sample ID of 431 isolates with AMR genes detected by WGS are reported in Additional file
4: Table S4. The combined WGS data of colistin-resistant
E. coli isolates will be further analysed via de novo assembly. ESBL genes were common among the collected
E. coli isolates and WGS analysis further identified
blaNDM-4 in 1 isolate,
blaNDM-5 in 6 isolates, and
blaKPC-2 in 1 isolate. Notably, of the 178
E. coli isolates, 151 isolates carried at least one of the five common CTX-M genes:
blaCTX-M group 1 (
blaCTX-M-15,
blaCTX-M-55) or
blaCTX-M group 9 (
blaCTX-M-14,
blaCTX-M-24, and
blaCTX-M-27). Three isolates carried both
blaCTX-M group 1 and group 9 genes. Forty-two isolates carried
blaOXA-1. Thirteen isolates carried AmpC β-lactamase
blaCMY. Four isolates carried AmpC β-lactamase
blaDHA (Table
4). Of note, the 16S methyltransferase
rmtB, which confers resistance to all clinically relevant aminoglycosides, was identified in two isolates. Six
E. coli isolates carried the colistin resistant genes:
mcr-1,
mcr-3, or both
mcr-1 and
mcr-3 (Table
4). The six isolates also harbored
blaCTX-M-55.
WGS data analysis of 102 K
. pneumoniae isolates identified 80 antibiotic resistance genes, including the carbapenemases
blaNDM-1,
blaNDM-5, variants of
blaOXA-48-like, and 1 isolate carried
blaIMP-15. The majority of
K. pneumoniae isolates carried a variant of the CTX-M genes, with 91 out of 102 carrying
blaCTX-M-15. One
K. pneumoniae isolate carried
blaCTX-M-15 and
blaCTX-M group 8/25, which was identified to be
blaCTX-M-63 by WGS. Of particular concern was the high prevalence of the carbapenemase genes identified in
K. pneumoniae (48/102), compared to other Gram-negative species, with both
blaNDM-1 and one of the three
blaOXA-48-like variants:
blaOXA-181,
blaOXA-232, and
blaOXA-247 identified. A number of isolates carried multiple aminoglycoside modifying enzymes (AMEs) that confer resistance to gentamicin and tobramycin but no 16S methyltransferase were identified. One
K. pneumoniae carried the colistin resistant gene,
mcr-1 and
blaCTX-M-55 (Table
4).
Based on the antibiotic resistance profile, 93 out of 97
A. baumannii strains carried a combination of carbapenem-resistant genes with 93 isolates harboring the most prevalent Class D carbapenemase,
blaOXA-23, which has been reported to be associated with carbapenem resistance
A. baumannii worldwide [
30]. Two isolates also carried an additional
blaOXA-58, a plasmid-borne resistance gene which has been shown to contribute significantly to carbapenem resistance in
A. baumannii [
31]. Four isolates carried the potent carbapenemase resistance gene,
blaNDM-1 (Table
4). Notably, 71 isolates also carried
armA (aminoglycoside resistance methylase), which confers resistance to all clinically relevant aminoglycosides.
WGS analysis of
P. aeruginosa identified 59 antibiotic resistance genes. Two carbapenemase genes were also detected in 3 isolates, 1 isolate carried
blaNDM and 2 isolates carried
blaVIM genes (Table
4). Two isolates also carried a gene encoding for the 16S methyltransferase,
rmtE, which confers resistance to aminoglycosides. ESBL producing strains were also identified, with 5 isolates carrying
blaVEB (Table
4).
The 4
Enterobacter spp. isolates were identified to be
E. aerogenes (current name:
Klebsiella aerogenes [
32]
, 1 isolate),
E. cloacae (1 isolate), and
E. hormaechei (2 isolates). The
E. aerogenes isolate carried
blaNDM-1. Only
blaCTX-M-15 was identified in the remaining
Enterobacter spp. (Table
4). Although the
Enterobacter spp. in this study do not carry a large number resistance genes, they harbor genes that confer resistance to clinically significant antibiotic classes.
E. faecium isolates identified by
ddlE gene were confirmed as
E. faecium by WGS, which revealed 18 different loci of antibiotic resistance genes including the six genes that comprise the vancomycin resistance operon (
vanS,
vanR,
vanH,
vanA,
vanB and
vanX) [
33].
S. aureus isolates (21/25, 84%) were positive for the
mecA gene indicating a significant presence of MRSA in studied isolates (Table
4). From WGS analysis,
qacA and
qacB genes, conferring tolerance to quaternary ammonium disinfectants, were identified in 4 and 5 MRSA isolates, respectively. Moreover, 1 MRSA isolate also carried the
mupA gene, which is involved in high-level resistance to mupirocin, (Table
3).