Molecular Analysis of Carbapenem and Aminoglycoside Resistance Genes in Carbapenem-Resistant Pseudomonas aeruginosa Clinical Strains: A Challenge for Tertiary Care Hospitals

Carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa) strains have become a global threat due to their remarkable capability to survive and disseminate successfully by the acquisition of resistance genes. As a result, the treatment strategies have been severely compromised. Due to the insufficient available data regarding P. aeruginosa resistance from Pakistan, we aimed to investigate the resistance mechanisms of 249 P. aeruginosa strains by antimicrobial susceptibility testing, polymerase chain reaction for the detection of carbapenemases, aminoglycoside resistance genes, extended-spectrum beta-lactamases (ESBLs), sequence typing and plasmid typing. Furthermore, we tested silver nanoparticles (AgNPs) to evaluate their in vitro sensitivity against antimicrobial-resistant P. aeruginosa strains. We observed higher resistance against antimicrobials in the general surgery ward, general medicine ward and wound samples. Phenotypic carbapenemase-producer strains comprised 80.7% (201/249) with 89.0% (179/201) demonstrating genes encoding carbapenemases: blaNDM-1 (32.96%), blaOXA48 (37.43%), blaIMP (7.26%), blaVIM (5.03%), blaKPC-2 (1.12%), blaNDM-1/blaOXA48 (13.97%), blaOXA-48/blaVIM (1.68%) and blaVIM/blaIMP (0.56%). Aminoglycoside-modifying enzyme genes and 16S rRNA methylase variants were detected in 43.8% (109/249) strains: aac(6′)-lb (12.8%), aac(3)-lla (12.0%), rmtB (21.1%), rmtC (11.0%), armA (12.8%), rmtD (4.6%), rmtF (6.4%), rmtB/aac(3)-lla (8.2%), rmtB/aac(6′)-lla (7.3%) and rmtB/armA (3.6%). In total, 43.0% (77/179) of the strains coharbored carbapenemases and aminoglycoside resistance genes with 83.1% resistant to at least 1 agent in 3 or more classes and 16.9% resistant to every class of antimicrobials tested. Thirteen sequence types (STs) were identified: ST235, ST277, ST234, ST170, ST381, ST175, ST1455, ST1963, ST313, ST207, ST664, ST357 and ST348. Plasmid replicon types IncFI, IncFII, IncA/C, IncL/M, IncN, IncX, IncR and IncFIIK and MOB types F11, F12, H121, P131 and P3 were detected. Meropenem/AgNPs and Amikacin/AgNPs showed enhanced antibacterial activity. We reported the coexistence of carbapenemases and aminoglycoside resistance genes among carbapenem-resistant P. aeruginosa with diverse clonal lineages from Pakistan. Furthermore, we highlighted AgNP’s potential role in handling future antimicrobial resistance concerns.


Introduction
Pseudomonas aeruginosa (P.aeruginosa) strains exhibit exceptional environmental adaptability due to the larger genome making it competent for massive metabolic flexibility, encoding several virulence factors and extensive efflux pump system.P. aeruginosa is known as a prominent bacterium involved in healthcare-associated nosocomial infections [1][2][3].Therapeutic options are severely compromised due to the exploitation of acquired and intrinsic resistance mechanisms by multidrug-resistant (MDR) P. aeruginosa [2].For instance, MDR P. aeruginosa strains appeared more efficiently when different classes of antimicrobials were used sequentially [4].However, the complexities of the underlying resistance mechanisms of MDR P. aeruginosa do not neatly correlate with the presence of resistance determinants nor with antimicrobial treatment regimes in clinical settings [4,5].P. aeruginosa core genome carries several transferrable resistance determinants, especially genes encoding for class B carbapenemases or ESBLs, fluoroquinolones and aminoglycoside-modifying enzymes (AMEs) [6][7][8].Carbapenem-resistant P. aeruginosa (CRPA) strains are considered a major health threat due to a high mortality rate of 20-30% [9,10].The Antimicrobial Testing Leadership and Surveillance (ATLAS) program recorded approximately 20% CRPA prevalence during 2008 to 2018 [11].The Chinese Antimicrobial Surveillance Network (CHINET) described 25-30% CRPA prevalence from 2005 to 2018 to 20-24% from 2019 to 2021 [12].The enduring issue of the emergence of carbapenem resistance among P. aeruginosa has been growing exponentially over time with a significant global prevalence [12][13][14][15][16]. Therefore, CRPA was classified as one of the three critical priority pathogens by the World Health Organization that demands urgent antimicrobial interventions due to exhausted reserves of antibiotics [17].
The evolution of epidemic clones jeopardizes all efforts to manage P. aeruginosa infections.One of the possible strategy to manage resistance is to use combination treatment, especially with non-antibiotic drugs that may interact with antibiotics [55,56].In this context, a prime non-antibiotic treatment is the use of silver nanoparticles (AgNPs) with powerful bactericidal properties against MDR microbes [57].AgNPs facilitate antimicrobial uptake into bateria and lower the desired antibiotic dose required resulting in enhanced antimicrobial activity [58].The antimicrobial response of AgNPs against MDR P. aeruginosa is evident from several reports [59][60][61]; however, CRPA has not been studied before.
Given the little information available from Pakistan regarding the contribution of different mechanisms in resistance development among P. aeruginosa strains, we aimed to study the local circulation of carbapenemases, AMEs and RMT resistance genes and the molecular mechanisms that are responsible for rapidly evolving CRPA isolates from Pakistan.Furthermore, we investigated the effect of AgNPs on the in vitro antimicrobial activity of carbapenems and aminoglycosides against CRPA clinical isolates.

Characteristics of Bacterial Strains and Antimicrobial Resistance Profile
Carbapenem-resistant clinical strains of P. aeruginosa (CRPA) were collected from different tertiary healthcare facilities in Lahore from 20 March 2022 to 11 April 2023.Out of 249 strains, 54.2% (n = 135) were obtained from female patients while 45.8% (n = 114) were from males.The clinical strains were retrieved from different clinical wards, sampling sources and age groups (Figure 1A-C).Antimicrobial susceptibility profile showed higher resistance against commonly used antimicrobials as shown in Figure 1D.
Given the little information available from Pakistan regarding the contribution of different mechanisms in resistance development among P. aeruginosa strains, we aimed to study the local circulation of carbapenemases, AMEs and RMT resistance genes and the molecular mechanisms that are responsible for rapidly evolving CRPA isolates from Pakistan.Furthermore, we investigated the effect of AgNPs on the in vitro antimicrobial activity of carbapenems and aminoglycosides against CRPA clinical isolates.

Characteristics of Bacterial Strains and Antimicrobial Resistance Profile
Carbapenem-resistant clinical strains of P. aeruginosa (CRPA) were collected from different tertiary healthcare facilities in Lahore from 20 March 2022 to 11 April 2023.Out of 249 strains, 54.2% (n = 135) were obtained from female patients while 45.8% (n = 114) were from males.The clinical strains were retrieved from different clinical wards, sampling sources and age groups (Figure 1A-C).Antimicrobial susceptibility profile showed higher resistance against commonly used antimicrobials as shown in Figure 1D.The pattern of antimicrobial resistance dissemination in relation to hospital wards and specimen type demonstrated that higher resistance against antimicrobials was observed in wound samples while higher resistance of ciprofloxacin (CIP) and gentamycin (GEN) was observed in the general surgery ward and general medicine ward, respectively.The results are shown in Figure 2.
Ceftazidime; FEP, Cefepime; FOS, Fosfomycin; AK, Amikacin; GEN, Gentamycin; CIP, Ciprofloxacin; PB, Polymyxin B; TZP, Piperacillin Tazobactam The pattern of antimicrobial resistance dissemination in relation to hospital wards and specimen type demonstrated that higher resistance against antimicrobials was observed in wound samples while higher resistance of ciprofloxacin (CIP) and gentamycin (GEN) was observed in the general surgery ward and general medicine ward, respectively.The results are shown in Figure 2.

Antimicrobial Efficacy of Silver Nanoparticles
P. aeruginosa clinical strains coharboring carbapenem and aminoglycoside resistance genes (n = 77) were used to check the antimicrobial efficacy of AgNPs in combination with MEM and AK separately.The bacterial cultures were grown in the presence of meropenem (MEM), amikacin (AK), AgNPs, MEM/AgNPs and AK/AgNPs, and MIC values were recorded.Higher MIC values were noted in the bacterial growth when cultured alone in the presence of MEM, AK and AgNPs.However, a reduction in MIC values was observed in the presence of MEM/AgNPs and AK/AgNPs.The results are given in Table 3.

Discussion
Antimicrobials' misuse has resulted in resistance development at an alarming rate against commonly used drugs [62,63].Patients with resistant P. aeruginosa infections have poorer prognoses, hence constant monitoring is crucial [64].Due to the lack of data from Pakistan, we herein collected data on CRPA resistance determinants and analyzed the antimicrobial activity of AgNPs.
Our data revealed that the main reservoirs of CRPA were the general surgery ward, general medicine ward and ICU, while the ICU was reported as the main recovery site of CRPA previously [3,73,80,84].Urine and wound samples were observed to be the common source of CRPA infection as observed previously [73,85].However, other sources reported including blood, cerebrospinal fluid, respiratory tract, musculoskeletal and genitourinary infections [3,14,18].The predominant age group in our study was 31-40 years.Our data are in contrast with previously reported age groups such as 41-60 years from Pakistan [72], >60 years of age from China [12,79], 46-60 years from India [86] and 45-65 years from England [87].It is evident from these reports that CRPA infections are frequently reported in old age group; however, its incidence among age groups 31-45 years and <20 years has been described in India and Oman [86,88].
Non-carbapenemase-producing carbapenem resistance mechanisms have been more prevalent among P. aeruginosa; however, the current emergence of carbapenemases is playing a critical role in resistance development [18,20,[89][90][91].It has been reported that carbapenemase-producing CRPA infections are associated with higher mortality rates than non-carbapenemase-producing CRPA infections [18].Our analysis demonstrated 80.7% carbapenemase producer CRPA isolates, which is higher as compared to global reports such as 25.07% and 33% from 17 health facilities in 12 countries [90,91].However, variable rates are reported in Pakistan: 18.4% and 52% [21,92].The variability observed among carbapenemase-producing CRPA prevalence might be due to the deficient testing efforts for detecting P. aeruginosa carbapenemase production suggesting a possible higher prevalence than reported [91].
In this regard, AgNPs earned attention due to their antimicrobial activity with efficient cell membrane penetration [146].Antimicrobial-loaded nanoparticles have been extensively used for the inhibition of P. aeruginosa infections previously [147].In vitro studies have proved the significant antimicrobial effects on P. aeruginosa isolates with effective growth inhibition [146,148].Specifically, the antimicrobial and nanoparticle combination proved to enhance antimicrobial efficacy, such as AMP/AgNPs possessing better killing efficiency of ampicillin-resistant P. aeruginosa isolates [2,149,150].We observed a significant reduction in the MIC values of CRPA in the presence of MEM/AgNPs and AK/AgNPs.

Conclusions
Our study contributed to understanding the antimicrobial resistance pattern existing among P. aeruginosa clinical isolates from Pakistan.We described the coexistence of carbapenemases and aminoglycoside resistance genes among CRPA with diverse clonal lineages from Pakistan for the first time.Furthermore, augmented antimicrobial activity of MEM/AgNPs and AK/AgNPs was identified, highlighting AgNPs' potential role in handling future AMR issues.Therefore, constant monitoring efforts are warranted to develop effective strategies for the control of CRPA and to reduce the incidence of untreatable infections in clinical settings.

Sampling and Identification of Clinical Strains
During the period of 20 March 2022 to 11 April 2023, a total of 249 clinical strains of carbapenem-resistant P. aeruginosa were identified and collected from patients who attended different tertiary healthcare facilities in Lahore, Punjab, Pakistan.Strains were phenotypically characterized by analyzing colony morphology and Grams's staining by culturing on MacConkey agar and cysteine lactose electrolyte-deficient media (Oxoid Ltd., Basingstoke, UK) for urine samples.Strains were biochemically characterized by API-20NE (BioMerieux, Marcy-IEtoile, France).
Modified carbapenem inactivation method (mCIM) was used to determine carbapenemase production by bacterial strains [152].In brief, 2 to 3 bacterial growth colonies were mixed with 2 mL of tryptone soy broth (TSB media; ThermoFischer Scientific, Waltham, MA, USA).Under sterile conditions, MEM disc (10 µg) was added into the bacterial suspension and incubated at 35 • C ± 2 • C for 4 h.Meantime, mCIM indicator strain suspension (carbapenem-sensitive strain; E. coli ATCC 25922) was prepared at a turbidity equivalent to 0.5 McFarland and inoculated on MHA (Oxoid, UK) plate.After 4 h incubation of bacterial strain in TSB media, the MEM disc was transferred to inoculate the MHA plate with indicator strain.Quality control strain K. pneumoniae ATCC BAA-1705 was used.The plate was incubated for 18 to 24 h at 35 • C ± 2 • C. CHROMagar TM ESBL media (CHROMagar, Paris, France) was used to identify ESBL-producer strains.

Molecular Identification of P. aeruginosa and Antibiotic Resistance Genes Detection
Genomic DNA was extracted from bacterial cultures by heat lysis method [153].Briefly, 500 µL sterile dH 2 O was taken in a 1.5 mL microcentrifuge tube and 3-5 bacterial colonies were added to it.Samples were mixed by vortexing for a few seconds.Incubation of bacterial colonies was performed at 98 • C for 10 min at 300 rpm in a thermomixer (Fischerscientific, Waltham, MA, USA).The sample was centrifuged at 1000 rpm for 10 min and the supernatant containing DNA was collected in a new tube.DNA was stored at −80 • C until further processing.The molecular identification of P. aeruginosa was performed by polymerase chain reaction (PCR) of 16S rDNA-based primers as described before [154].Standard PCR was used to detect carbapenem resistance-encoding genes (bla NDM-1 , bla OXA-48 , bla KPC-2 , bla VIM and bla IMP ), ESBLs (bla SHV , bla TEM and bla CTX-M ) and aminoglycoside resistance genes (aac(6 ′ )-lb, aac(3)-lla, rmtB, rmtC, armA, rmtD, rmtF) [73].Genomic DNA was amplified in 50 µL reaction volume containing 25 µL of 2x PCR Master Mix (catalog # K0171, Thermoscientific, Waltham, MA, USA), 10 pM of each primer, 300 ng of DNA and dH 2 O up to 50 µL in a thermal cycler (Proflex PCR system, Thermo Fischer Scientific, Waltham, MA, USA).Amplicons were resolved and analyzed by agarose gel electrophoresis (1-1.5%)stained with Syber TM Safe DNA gel stain (catalog # S33102, Thermoscientific, Waltham, MA, USA) and gel documentation system (G:BOX iChemiXT, Syngene, Cambridge, UK).The primer sequences and PCR cycling conditions are given in Table 4.

Determination of bla NDM and bla KPC Alleles
For bla NDM and bla KPC allele determination, Sanger's sequencing method was applied by using the BigDye Terminator v3.1 kit for cycle sequencing as per kit recommendations.Cycle sequencing PCR was carried out in 10 µL PCR reaction volume containing 4 µL BigDye terminator 3.1 Ready Reaction Mix, 0.5 µL (3.2 pmol) forward primer, 2 µL purified DNA template (5-20 ng) and 3.5 µL dH 2 O.The following PCR cycling conditions were used: 96 • C 1 min, 96 • C 10 s, 50 • C 5 s, 60 • C 2 min (35 cycles).Purification of PCR product was carried out by using the BigDye XTerminator purification kit.The capillary electrophoresis was carried out by Genetic Analyzer (ABI-3500, Thermo Fischer, Waltham, MA, USA).Sequencing analysis software v6.1 and basic local alignment tools (BLAST, NCBI) were used for data analysis and interpretation.CLC Sequence Viewer 7 version 7.0.2 was used for sequence alignment and mutation analysis.
Plasmid DNA was extracted from a single colony of P. aeruginosa by using the plasmid isolation kit (ThermoFischer Scientific, Waltham, MA, USA).Plasmid classification was performed according to their incompatibility groups by using the PCR-based replicon typing (PBRT) method as described previously [157].Furthermore, Degenerate Primer MOB Typing was used for the classification of γ-proteobacterial transmissible plasmids in five phylogenetic relaxase MOB families (MOB F , MOB P , MOB Q , MOB H and MOB C ) [158].

Evaluation of Antimicrobial Activity of AgNPs
The broth microdilution checkerboard method was used to evaluate the antimicrobial activity of AgNPs, MEM and AK against the P. aeruginosa strains coharboring carbapenem and aminoglycoside resistance genes.AgNPs were purchased from Sigma (Cat # 730785, Sigma-Aldrich, St. Louis, MO, USA).The particle size of AgNPs was 10 nm with a solution concentration of 20 µg/mL in aqueous buffer containing sodium citrate as a stabilizer.MEM, AK and AgNP dilutions were prepared in Mueller Hinton broth, and bacterial cultures were prepared at a concentration of 0.5 McFarland (10 8 CFU/mL) and further diluted to 1:100 to reach the final concentration of 10 6 CFU/mL.In a sterile 96-well microtiter plate, each well was inoculated with 100 µL of diluted bacterial suspension and mixed with antibiotic solution.All tests were conducted in duplicate with a growth control without the addition of antibiotics and with sodium citrate addition.The inoculated microtiter plate was incubated at 37 • C for 18 h.After incubation, the fractional inhibitory concentration index (∑FIC) was calculated by dividing the individual MIC of treatments by MIC of the combination drugs.∑FIC value lower than 0.5 showed synergistic effect, values between 0.5 and 4.0 indifferent and values above 4 antagonistic effect [159].

Table 4 .
Primers used for PCR and sequencing.