This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee, Shenzhen Children’s Hospital, reference number: 2018 (013) dated 2018/09/03, which complies with international ethical standards. All experiments were conducted as per the hospital biosafety regulations act. All P. aeruginosa isolates were collected between Feb 1, 2021, to Feb 28, 2022, from diverse clinical samples such as blood, urine, pus, stool and pleural secretion received at the central microbiology laboratory of Shenzhen Children’s Hospital, Shenzhen, as part of routine hospital investigation. Only verbal consent was obtained because no personal information was used for research purposes, therefore written consent was not required.
Clinical sampling and identification of isolates
A total of 294 non-repetitive isolates were collected, and other electronic data including gender, age, hospitalized department and treatment of antibiotics were extracted from the medical record room. P. aeruginosa was cultured on blood agar and identified using the VITEK®2 compact system (BioMerieux, France) and confirmed by the API-20 and 16S-RNA sequencing (Sangan Biotechnology, Shanghai). The genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s instructions. The quality of extracted DNA was assessed by measuring the absorbance at 260 and 280 nm wavelengths. The cultures were kept for 3 days (SD 2.83) followed by storage in 30% glycerol stock at -80°C for further investigation. The full length of the 16S rRNA gene was amplified by conventional PCR by using universal primers as per the protocol described earlier [20]. PCR reaction total volume was 20 µL containing 1µL (30 ng) of genomic DNA, 10 µL of 2X Master Mix, 0.4 µL (20 pmol) of each forward and reverse primer and 8.2 µL of nuclease-free water. Thermocycler conditions were set at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. Deionised sterile distilled water was used as the control while P. aeruginosa ATCC 27853 DNA was used as a positive control. All amplified products were run on 1.8% agarose gel and stained with ethidium bromide. The products were purified using the QIAquik gel extraction kit as per manufacturer instructions. The purified products were sequenced (forward and backwards) by Sangon Biotech, Shanghai. The sequenced results were aligned with published reference sequences in the NCBI BLAST database and confirmed as P. aeruginosa. All statistical analyses were performed using Chi-square 2-tailed test, and P values <0.05 were considered significant.
Antimicrobial susceptibility and characterization of resistance genes
The antimicrobial susceptibility test (AST) was performed on all 294 confirmed isolates using the VITEK®2 compact system (BioMerieux, France) with the standard AST09 card (software version 9.01). The antimicrobial agent tested included aminoglycosides (gentamicin, amikacin, tobramycin), carbapenems (imipenem, meropenem), and cephalosporins (ceftazidime, cefepime), fluoroquinolones (ciprofloxacin, levofloxacin), penicillins plus β-lactamase inhibitors (piperacillin-tazobactam), monobactams (aztreonam). Isolates showing intermediate antimicrobial-resistant strains were considered resistant strains for statistical analysis [21]. The AST results were accurately interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints 2017 [22]. A positive control strain characterized by our laboratory was used in addition to P. aeruginosa ATCC27835 as a control strain. The ESBL production was initially determined by using the VITEK®2 compact system (BioMerieux, France) and confirmed by the double-disc diffusion method (DDDM) which includes a disc of amoxicillin-clavulanic acid (20/10 mcg), ceftriaxone (30 mcg), ceftazidime (30 mcg) and cefotaxime (30 mcg) [23]. A Salmonella enterica ST34 strain SP-15-127 was used as a positive control strain which had been previously characterized in our laboratory as a strain containing blaCTX-M-15 ESBL gene [20], and E. coli ATCC25922 was used as a negative control. The ESBLs production results were analyzed according to the Clinical and Laboratory Standards Institute (CLSI-2017) guidelines [22]. Isolates classified as β-lactamase producers (n=126) were tested by the PCR assay to determine the β-lactamase encoding genes such as blaVIM, blaKPC, blaNDM, blaCTX-M, blaOXA-48 and blaIMP, aminoglycoside resistance gene aac(3)IIIa and glycylcycline resistance tatA(Aa) were screened for with primers previously described and listed in (Supplementary Table 1) [24]. DNA sequencing was performed at Sangon Biotech Pvt Ltd, Shanghai on the amplification of PCR-positive products, and the obtained results were compared and precisely aligned with published reference sequences using the online NCBI BLAST database to identify any specific resistant genes.
Molecular typing
To uniquely determine the clonal relationship between the P. aeruginosa strains, we performed strain-typing based on seven housekeeping genes acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE according to the instructions on MLST strain typing website for P. aeruginosa (http://pubmlst.org/paeruginosa/). The primers for the amplification of the above housekeeping genes were adopted as described earlier by Curran et al [25] (Supplementary Table 2) PCR conditions were developed in our laboratory with a 20 µl amplification reaction mixture comprised of 1µL (30 ng) of genomic DNA, 10 µL of 2X Master Mix (Sangaon Biotech), 0.4 µL (20 pmol) of each forward and reverse primer and 8.2 µL of nuclease-free water. Thermocycler conditions were set at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. Deionised sterile distilled water was used as the control while P. aeruginosa ATCC 27853 DNA was used as a positive control. All PCR products run through 1.5% agarose gel with ethidium bromide and specific product were purified using the gel extraction kit (QIAquik gel extraction Kit) as per manufacturer instructions. The purified products were sequenced by Sangon Biotech, Shanghai. The sequences were analysed using the MLST database (https://pubmlst.org/paeruginosa) and sequence type (ST) was determined. The combinations of alleles which did not yield a match were designated as a “new” ST. The ST types and antimicrobial drug resistance group correlation were established by using Gephi network analysis software. Furthermore, we fully analysed the possible distribution of the resistance encoding genes among the 126 isolates of ESBLs producing P. aeruginosa justifiably concerning STs type. The data were visualised by PRISMA 8.0 software.
Plasmid Typing
Plasmid DNA was isolated using the Invitrogen PureLink-HiPure plasmid filter miniprep standard kit as per manufacturer instructions. The Plasmid incompatibility (Inc) group were determined by typically using PCR-based replicon typing (PBRT). Eighteen specific primer pairs were designed, based on the multiple comparative analysis of nucleotide sequence on the EMBL Gene Databank, for HI1, HI2, I1-Iγ, X, L/M, N, FIA, FIB, W, Y, P, FIC, A/C, T, FIIAS, F, K and B/O replicons and results analysed as described earlier [26]. Amplicons were visualized by gel electrophoresis on a 1.8% agarose gel stained with ethidium bromide. Genomic DNA of E. coli DH5α strain was used as the negative control. The degenerate primer MOB typing (DPMT) was detected by using amplification of the 19 key pairs of primers which were encoded in 33 reference MOB relaxase genes. The primers and PCR assay protocol were as described earlier [27]. All primers have been listed in Supplementary Table 2