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.

Association of Surfactant Protein D Single Nucleotide Polymorphisms rs721917, rs2243639, rs3088308 with Recurrent Aphthous Stomatitis in Pakistani Population.

Recurrent aphthous stomatitis (RAS) is a benign ulcerative condition, defined by the recurrent formation of non-contagious mucosal ulcers. Surfactant protein D (SP-D) is secreted frequently at surfaces exposed directly to body fluids. This study aims to investigate the association of SP-D single nucleotide polymorphisms (SNPs) with the onset of RAS. Blood samples from 212 subjects (106 cases/controls each) were collected during 2019 and genotyped for SP-D SNPs (rs721917, rs2243639, rs3088308) by polymerase chain reaction and restriction fragment length polymorphism followed by 12% polyacrylamide gel electrophoresis. Minor aphthous (75.5%) was the commonly observed ulcer type as compared to herpetiform (21.7%) and major aphthous ulcers (2.8%). A family history of RAS was reported in 70% of cases. RAS was found significantly associated with rs3088308 genotypes T/A (95% (Cl): 1.57-5.03, p = 0.0005), A/A (95% (Cl): 1.8-6.7, p = 0.0002), T-allele (95% (Cl): 1.09-2.36, p = 0.01), A-allele (95% (Cl): 1.42-3.91, p = 0.01), rs721917 genotype T/T (95% (Cl): 1.15-25.35, p = 0.03), and T-allele (95% (Cl): 1.28-3.10, p = 0.002). Female gender and obese body mass index (BMI) were significantly associated with rs3088308 genotypes T/A (95% (CI): 1.89-15.7, p = 0.001), T/T (95% (Cl): 1.52-11.9, p = 0.005), A-allele (95% (Cl): 1.65-7.58, p < 0.001), and T-allele (95% (Cl): 1.4-10.1, p <0.001) and rs721917 genotype T/T (95% (CI) = 1.3-33, p = 0.02), respectively. This study describes the association of SP-D SNPs (rs721917, rs3088308) with RAS in the Pakistani population.

Estimation, Evaluation and Characterization of Carbapenem Resistance Burden from a Tertiary Care Hospital, Pakistan.

Carbapenem resistance has become major concern in healthcare settings globally; therefore, its monitoring is crucial for intervention efforts to halt resistance spread. During May 2019-April 2022, 2170 clinical strains were characterized for antimicrobial susceptibility, resistance genes, replicon and sequence types. Overall, 42.1% isolates were carbapenem-resistant, and significantly associated with Klebsiella pneumoniae (K. pneumoniae) (p = 0.008) and Proteus species (p = 0.043). Carbapenemases were detected in 82.2% of isolates, with blaNDM-1 (41.1%) associated with the ICU (p < 0.001), cardiology (p = 0.042), pediatric medicine (p = 0.013) and wound samples (p = 0.041); blaOXA-48 (32.6%) was associated with the ICU (p < 0.001), cardiology (p = 0.008), pediatric medicine (p < 0.001), general surgery (p = 0.001), general medicine (p = 0.005) and nephrology (p = 0.020); blaKPC-2 (5.5%) was associated with general surgery (p = 0.029); blaNDM-1/blaOXA-48 (11.4%) was associated with general surgery (p < 0.001), and wound (p = 0.002), urine (p = 0.003) and blood (p = 0.012) samples; blaOXA-48/blaVIM (3.1%) was associated with nephrology (p < 0.001) and urine samples (p < 0.001). Other detected carbapenemases were blaVIM (3.0%), blaIMP (2.7%), blaOXA-48/blaIMP (0.1%) and blaVIM/blaIMP (0.3%). Sequence type (ST)147 (39.7%) represented the most common sequence type identified among K. pneumoniae, along with ST11 (23.0%), ST14 (15.4%), ST258 (10.9%) and ST340 (9.6%) while ST405 comprised 34.5% of Escherichia coli (E. coli) isolates followed by ST131 (21.2%), ST101 (19.7%), ST10 (16.0%) and ST69 (7.4%). Plasmid replicon types IncFII, IncA/C, IncN, IncL/M, IncFIIA and IncFIIK were observed. This is first report describing the carbapenem-resistance burden and emergence of blaKPC-2-ST147, blaNDM-1-ST340 and blaNDM-1-ST14 in K. pneumoniae isolates and blaNDM-1-ST69 and blaNDM-1/blaOXA-48-ST69 in E. coli isolates coharboring extended-spectrum beta-lactamases (ESBLs) from Pakistan.

Characterization of Genomic Diversity among Carbapenem-Resistant Escherichia coli Clinical Isolates and Antibacterial Efficacy of Silver Nanoparticles from Pakistan.

The emergence of carbapenem-resistant Escherichia coli (E. coli) is considered an important threat to public health resulting in resistance accumulation due to antibiotics misuse and selection pressure. This warrants periodic efforts to investigate and develop strategies for infection control. A total of 184 carbapenem-resistant clinical strains of E. coli were characterized for resistance pattern, resistance genes, plasmids, sequence types and in vitro efficacy of silver nanoparticles (AgNPs). Carbapenem resistance was prevalent in E. coli isolated from female patients (64.7%), urine samples (40.8%) and surgical wards (32.1%). Polymyxin-B showed higher susceptibility. ESBLs and carbapenemases were produced in 179 and 119 isolates, respectively. Carbapenemase-encoding genes were observed among 104 strains with blaNDM-1 (45.1%), blaOXA-48 (27%), blaNDM-7 (3.8%), blaNDM-1/blaOXA-48 (15.4%), blaNDM-7/blaOXA-48 (2.9%), blaOXA-48/blaVIM (3.8%) and blaNDM-1/blaVIM (2%). ESBL resistance genes were detected in 147 isolates, namely blaSHV (24.9%), blaCTX-M (17.7%), blaTEM (4.8%), blaSHV/blaCTX-M (29.2%), blaSHV/blaTEM (15%) and blaCTX-M/blaTEM (8.8%). ST405 (44.4%) and ST131 (29.2%) were more frequent sequence types with ST101 (9.7%), ST10 (9.7%) and ST648 (7%). The replicon types IncFII, IncFIIK, IncA/C, IncN and IncL/M were detected. The combination of MEM/AgNPs remained effective against carbapenemase-positive E. coli. We reported genetically diverse E. coli strains coharboring carbapenemases/ESBLs from Pakistan. Moreover, this study highlights the enhanced antibacterial activity of MEM/AgNPs and may be used to manage bacterial infections.

Novel Carbapenem-Resistant Klebsiella pneumoniae ST147 Coharboring bla NDM-1, bla OXA-48 and Extended-Spectrum ß-Lactamases from Pakistan.

PURPOSE: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) is associated with the acquisition of multiple carbapenemases. Their clonal spread is a worldwide concern due to their critical role in nosocomial infections. Therefore, the identification of high-risk clones with antibiotic resistance genes is very crucial for controlling its global spread. MATERIALS AND METHODS: A total of 227 K. pneumoniae strains collected during April 2018 to November 2019 were confirmed by PCR. Carbapenemases and extended-spectrum ß-lactamases (ESBL) were detected phenotypically. Confirmation of carbapenemases was carried out by PCR and Sanger sequencing. The clonal lineages were assigned to selected isolates by multilocus sequence typing (MLST), and the plasmid analysis was done by PCR-based detection of the plasmid replicon typing. RESULTS: Of the total K. pneumoniae, 117 (51.5%) were carbapenem resistant (CRKP) and 140 (61.7%) were identified as ESBL producers. Intermediate to high resistance was detected in the tested ß-lactam drugs while polymyxin-B and tigecycline were found to be susceptible. Among CRKP, 91 (77.8%) isolates were detected as carbapenemase producing, while 55 (47%) were positive for bla NDM-1 23.9% (n=28), bla OXA-48 22.2% (n=26) and bla VIM 0.85% (n=1) while 12.7% (n=7) carried both bla NDM-1 and bla OXA-48 genes. The CRKP coharboring bla NDM-1 and bla OXA-48 genes (n=7) were positive for bla CTX-M bla SHV (n=3), bla SHV (n=1) and bla CTX-M (n=3). The novel CRKP with the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes were associated with the high-risk clone ST147 (n=5) and ST11 (n=2). The assigned replicon types were IncL/M, IncFII, IncA/C and IncH1. CONCLUSION: This is the first report of the coexistence of bla NDM-1, bla OXA-48, bla CTX-M and bla SHV genes on a high-risk lineage ST147 from Pakistan. This study highlights the successful dissemination of carbapenemase resistance genes in the high-risk clones that emphasizes the importance of monitoring and controlling the spread of these diverse clones globally.

Estimation of serum matrix metalloproteinases among patients of oral squamous cell carcinoma.

OBJECTIVE: To estimate the serum levels of matrix metalloproteinases in oral squamous cell carcinoma patients and in healthy subjects. METHODS: In this observational study, biopsy diagnosed oral squamous cell carcinoma patients (n= 38) were recruited from Mayo Hospital, Lahore during 2016 to 2017. Age and gender matched Controls (n= 38) were also included. Venous blood sample of each participant was drawn, serum separated and the levels of matrix metalloproteinases were measured by multiplex ELISA. RESULTS: Serum levels of MMP-1, -8, -10, -12 and -13 in OSCC patients showed statistically significant increase as compared to control group (p < 0.01). The MMP-12 predicted the presence of OSCC with highest AUC of 0.836 (95% CI [0.733 to 0.911]) for sensitivity and specificity of 80% and 78.9%, respectively for a cut-off value of 16.13 pg/ml. CONCLUSIONS: MMP-12 has been found to have significant sensitivity and specificity to qualify as a diagnostic biomarker.