Out of 5334 collected bacterial isolates, 320 (6%) corresponded to P. aeruginosa. The isolates were recovered from hospitalized patients with an average age of 59.72 years within the age range of 2 months and 88 years old. The clinical samples included lower respiratory tract samples (n = 120; 37.5%), wound specimens (n = 108; 33.75%), urine (n = 71; 22.2%), and blood (n = 21; 6.6%). Most of the P. aeruginosa isolates were obtained from the ICU (n = 178; 55.6%). Detailed information concerning specimen types, patient characteristics, and hospital units is listed in Table 1.
Table 1
Demographic and clinical characteristics of Pseudomonas aeruginosa-infected study patients
Characteristic | No. (%) |
Gender | |
Male gender | 172 (53.8) |
Female gender | 148 (46.2) |
Admission ward | |
Intensive care unit | 126 (39.4) |
Thoracic surgery | 3 (0.9) |
Orthopedic surgery | 3 (0.9) |
Plastic surgery | 6 (1.9) |
Cardiovascular surgery | 39 (12.2) |
Neurosurgery | 10 (3.1) |
General surgery | 29 (9.1) |
Urology surgery | 3 (0.9) |
Internal wards | 52 (16.3) |
Department for COVID-19-positive patients | 23 (7.2) |
Geriatric department | 19 (5.9) |
Department of Oncology | 7 (2.2) |
Type of specimen | |
Tracheal aspirate | 74 (23.1) |
Bronchoalveolar lavage | 26 (8.1) |
Sputum | 20 (6.3) |
Wound specimen | 108 (33.8) |
Blood | 21 (6.6) |
Urine | 71 (22.2) |
Comorbidity | |
Malignancy | 36 (11.2) |
Chronic venous insufficiency | 18 (5.6) |
Heart insufficiency | 18 (5.6) |
Diabetes | 23 (7.2) |
COVID-19 pneumonia | 23 (7.2) |
Invasive procedures | |
Any surgical procedure | 93 (29.1) |
Mechanical ventilation | 72 (22.5) |
Central venous catheter | 42 (13.1) |
Urinary catheter | 139 (43.4) |
Antimicrobial Resistance
The highest resistance prevalence (> 50%) was observed for fluoroquinolones, levofloxacin, and ciprofloxacin. Resistance prevalence against the tested beta-lactam antibiotics ranged from 25- 56.3%, being 43.1% for carbapenems (Fig. 2). The lowest resistance rates were observed for ceftazidime – avibactam, aztreonam, and colistin. Indeed, 99.7% of the isolates displayed in vitro susceptibility to colistin. All isolates had MIC values of colistin < 2 mg/L, except one isolate that had a MIC value > 16 mg/L.
Overall, 154 (48.1%) and 114 (35.6%) out of 320 P. aeruginosa isolates were MDR and XDR, respectively. The most common MDR phenotype observed was non-susceptibility to ceftazidime, piperacillin-tazobactam, ticarcillin, meropenem, levofloxacin, and ciprofloxacin (42%).
Among the 320 isolates of P. aeruginosa, 138 (43.1%) were CRPA, being resistant to meropenem and/or imipenem. The respective MICs were > 8 mg/L for meropenem and/or > 4 mg/L for imipenem. Of note, 23 out of 138 (16.7%) isolates of CRPA were susceptible to amikacin.
The frequency of CRPA isolates recovered from hospital wards (n = 138) was as follows: ICUs – n = 57 (41.3%), cardiovascular surgery – n = 32 (23.2%), general surgery – n = 10 (7.2%), neurosurgery – 5 (3.6%), orthopedic surgery – n = 4 (2.9%), plastic surgery – n = 2 (1.4%), internal medicine – n = 10 (7.2%%), geriatric department – n = 9 (6.5%), department for COVID-19-positive patients – n = 6 (4.3%), and department of oncology – n = 3 (2.2%).
The 138 CRPA isolates were identified as adequate candidates for the detection of the MBL-encoding genes.
Molecular Detection Of Mbl- And Esbl-encoding Genes
Among the 138 CRPA isolates, 31 (22.5%) harbored MBL-encoding genes, namely blaNDM−1 (Supplementary material Fig. 1). The highest number of MBL genes was identified in isolates from ICUs patients (n = 12; 38.7%), department for cardiovascular surgery (n = 9; 29.7%), or corona care centers (n = 5; 16.1%). The blaVIM and blaIMP genes were not identified in any of the tested isolates.
Only three of the 31 isolates (9.7%) found to be blaNDM-positive carried ESBL-encoding genes. Two isolates harbored simultaneously blaNDM−1 and blaPER−1, whereas one isolate carried blaNDM−1 and blaGES−5. Among the tested CRPA isolates, blaCTX−M, blaTEM, blaSHV, and blaVEB genes were not detected (Fig. 3).
Multilocus Sequence Typing Analysis
MLST analysis was performed for all blaNDM−1 positive P. aeruginosa isolates (n = 31). Among them, 25 and 6 isolates were classified as ST235 and ST654, respectively (Fig. 3). One isolate exhibiting resistance to all antimicrobial agents tested in the study, including colistin, belonged to ST235. In addition, two isolates co-harboring blaPER−1 and blaNDM−1 were assigned to ST235 (n = 1) and ST654 (n = 1), whereas the isolate harboring blaGES−5 gene was ST654 (n = 1). In summary, CRPA types identified in this study in Serbia were the following: ST235/blaNDM−1, ST235/blaNDM−1/blaPER−1, ST654/blaNDM−1, ST654/blaNDM−1/blaPER−1, and ST654/blaNDM−1/blaGES−5. Genotypes ST235/blaNDM−1 and ST654/blaNDM−1 were found in at least three different cities across Serbia, indicating dissemination throughout the country.
The geographic distribution of genomes and the antibiotic resistance profiles of genomes affiliated with ST235 and ST654 were assessed based on the genomes available in the Pseudomonas Genome Database (Fig. 4). From the public database were retrieved all available sequences and information of ST235 and ST654 P. aeruginosa isolates. The P. aeruginosa ST235 (n = 248) isolates presented a worldwide distribution, mostly in the United States of America (USA) (23% of the analyzed isolates). Serbia represented 10% of the ST235 isolates available in the online database (Fig. 4a). Instead, the ST654 (n = 18) isolates, although in a lower number, had mostly Serbia as the isolation country (39% of the isolates available in the online database) followed by the USA and Canada (11% of the isolates available in the online database) (Fig. 4b). Analyzing the antibiotic resistance genes profile in both ST235 and ST654 isolates were annotated different aminoglycoside resistance genes and carbapenemase resistance genes of different classes. Moreover, blaOXA−396 was observed in ST654 isolates, while blaOXA−488 gene was present in all genomes belonging to ST235 (Supplementary material Fig. S2).
The ST235 strains’ antibiotic resistance profile indicates the ARGs blaGES (31 isolates), blaNDM (30 isolates), and blaVIM (29 isolates) as the main carbapenem resistance genes harbored. Instead, ST654 isolates presented as main carbapenems resistance gene blaNDM (10 isolates) followed by blaGES (five isolates) and blaVIM (two isolates).
Phylogenomic analysis of blaNDM-positive strains
Details on genome sequencing and assembly quality of four randomly selected blaNDM-positive P. aeruginosa (NDM1_1, NDM1_2, NDM1_3, and NDM1_4) isolates can be found on the National Center for Biotechnology Information site, under BioProject Accession number PRJNA753000. Briefly, the genome sizes ranged 6.9-7.1Mbp, organized in 1–35 contigs, and coverages ranging from 100-300x, as shown in Supplementary Table S2.
The results of phylogenomic analysis of the four genomes of P. aeruginosa harboring the blaNDM gene together with 161 previously published genomes of the same STs with a known geographical location, available in the NCBI Pathogen Detection database (Supplementary Table S3.) are illustrated in Fig. 5.
Overall, the phylogenetic tree of the 165 P. aeruginosa isolates could be divided into two clades according to STs, as illustrated in Fig. 5. The ST235 isolates NDM1_2, NDM1_3, and NDM1_4 grouped were closely related to isolates from Croatia, Italy, USA, and Bulgaria, with detected SNP differences ranging from 59 to 1157 (Supplementary Table S4). The ST654 isolate NDM1_1 was grouped with isolates from Lebanon, India, Chile, and USA, with detected SNP differences ranging from 447 to 4048 (Supplementary Table S4).
The Genetic Context Of The Detected Mbl Genes
The genetic context of the three sequenced P. aeruginosa harboring blaNDM−1 gene have been analyzed and shown in Fig. 6. Because the blaNDM gene in NDM1_4 isolate was located at the very start of the assembled contig, its genetic context was not examined.
The blaNDM−1 gene of NDM1_1, NDM1_2, and NDM1_3 is contained within a class 1 integron-bearing insertion sequence (IS) common region 1 (ISCR1) (Fig. 6.). ISCR1 element is followed by the partial transposase gene ISAba14 (accession no. JQ080305.1), the aminoglycoside resistance gene aphA6, the transposase ISAba125 gene, the blaNDM−1 gene, the truncated bleomycin/qac resistance gene, and the sul1 end of a class 1 integron.
In NDM1_2 and NDM1_3 isolates, the region upstream of ISCR1 corresponds to a class 1 integron, with the intI1 gene followed by several gene cassettes: the genes aac(6’)Ii and aadA6 encoding the aminoglycoside-modifying enzymes AAC-6′(I) and AADA6, respectively, and DUF domain-containing protein. This last cassette is followed by a truncated qac/sul conserved sequence. Upstream of this complex class 1 integron, ISPa7 transposase was detected, as well as incomplete transposase belonging to the Tn3 family.
In the NDM1_1 isolate, the region upstream of ISCR1 also corresponds to a class 1 integron, with the intI1 gene, but is followed by a blaGES−5 gene and gene cassette sul1. Upstream of class 1 integron, the mercury resistance operon was detected between partial recombinase genes. In the region downstream of ISCR1, aminoglycoside resistance genes aph(6’)Id and aph(6’)Ib were identified, located between two Tn3 family transposases.
Of note, in all four genomes, the aminoglycoside resistance gene aphA6, encoding enzyme aminoglycoside 3′-phosphotransferase type VI (Aph(3′)-VI) conferring resistance to amikacin, was detected. In three analyzed genomes, it was located upstream of ISAba125 and blaNDM−1 gene. Other aminoglycoside resistance genes identified in four sequenced isolates include aadA6 (n = 3), aph(3’)-IIb (n = 3), aph(6’)Id (n = 1), aph(6’)Ib (n = 1).