Background Resistance to clarithromycin and levofloxacin in Helicobacter pylori which resulted in treatment failures has become a major challenge for physicians worldwide. The resistance is mainly mediated by mutations in a specific domain of the 23S rRNA, gyrA and gyrB genes for clarithromycin and levofloxacin respectively. Hence in this study, we aimed to investigate the current status of H. pylori resistance in our hospital to these two antibiotics based on the molecular approach. Materials and Methods Gastric biopsy samples were obtained from treatment-naïve patients. Bacterial genomic DNA was extracted using a commercial kit and continued with DNA amplification using polymerase chain reaction (PCR) with specific primers. The PCR amplicons were subjected to sequencing on 23S rRNA gene targeting nucleotide positions at 2,146, 2,147, 2,186 and amino acids at gyrA positions 87 and 91 and gyrB positions 436, 438, 481, 484 to investigate the possible mutations or polymorphisms of genes that lead to clarithromycin and levofloxacin resistance respectively. Results Sixty-one urease-positive gastric biopsy samples were studied. The findings revealed the primary resistance rates to clarithromycin was 14.8% and to levofloxacin was 3.3% in our current scenario based on detection of reported resistance-related mutations of A2147G and D91N in 23S rRNA and gyrA genes, respectively. Interestingly, we found a high rate of silent mutations of the gyrA codon 87Asn (32.8%, 20/61) and two polymorphisms of the gyrB D481E (16.4%, 10/61) and R484K (21.3%, 13/61). The role of these polymorphisms in gyrB remained to be elucidated whether the levels of levofloxacin resistance are related to the position/amino acid. Conclusion The primary resistance rate of H. pylori to clarithromycin has increased compared to the previous report in Malaysia. Therefore, molecular screening could aid and is important for the selection of antibiotics for H. pylori eradication therapies.

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Background Acinetobacter baumannii was reported to have resistance towards carbapenems and the ability to form an air-liquid biofilm (pellicle) which contributes to their virulence. The GacSA two-component system has been previously shown to play a role in pellicle formation. Therefore, this study aims to detect the presence of gacA and gacS genes in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates recovered from patients in intensive care units and to investigate their pellicle forming ability. Methods The gacS and gacA genes were screened in 96 clinical CRAB isolates using PCR assay. Pellicle formation assay was performed in Mueller Hinton medium and Luria Bertani medium using borosilicate glass tubes and polypropylene plastic tubes. The biomass of the pellicle was quantitated using the crystal violet staining assay. The selected isolates were further assessed for their motility using semi-solid agar and monitored in real-time using real-time cell analyser (RTCA). Results All 96 clinical CRAB isolates carried the gacS and gacA genes, however, only four isolates (AB21, AB34, AB69 and AB97) displayed the ability of pellicle-formation phenotypically. These four pellicle-forming isolates produced robust pellicles in Mueller Hinton medium with better performance in borosilicate glass tubes in which biomass with OD570 ranging from 1.984 ± 0.383 to 2.272 ± 0.376 was recorded. The decrease in cell index starting from 13 hours obtained from the impedance-based RTCA showed that pellicle-forming isolates had entered the growth stage of pellicle development. Conclusion These four pellicle-forming clinical CRAB isolates could be potentially more virulent, therefore further investigation is warranted to provide insights into their pathogenic mechanisms.

Acinetobacter baumannii forms air–liquid interface pellicles that boost its ability to withstand desiccation and increase survival under antibiotic pressure. This study aims to delve into the transcriptomic profiles of pellicle cells from clinical strains of carbapenem-resistant A. baumannii (CRAB). The total RNA was extracted from pellicle cells from three pellicle-forming CRAB strains and planktonic cells from three non-pellicle-forming CRAB strains, subject to RNA sequencing using Illumina HiSeq 2500 system. A transcriptomic analysis between pellicle and planktonic cells, along with differential expression genes (DEGs) analysis and enrichment analysis of annotated COGs, GOs, and KEGGs, was performed. Our analysis identified 366 DEGs in pellicle cells: 162 upregulated genes and 204 downregulated genes. The upregulated ABUW_1624 (yiaY) gene and downregulated ABUW_1550 gene indicated potential involvement in fatty acid degradation during pellicle formation. Another upregulated ABUW_2820 (metQ) gene, encoding the D-methionine transporter system, hinted at its contribution to pellicle formation. The upregulation of two-component systems, CusSR and KdpDE, which implies the regulation of copper and potassium ions in a CRAB pellicle formation was also observed. These findings provide valuable insights into the regulation of gene expression during the formation of pellicles in CRAB, and these are potential targets that may aid in the eradication of CRAB infections.