Exposure to antibiotics below their minimum inhibitory concentration (sub-MIC) is known to be a selection pressure for the development of antibiotic resistance. In this work, we aimed to provide more understanding of how antibiotic resistance and cross-resistance developed in Pseudomonas aeruginosa under sub-MIC exposure to Ciprofloxacin. Fully susceptible P. aeruginosa ATCC 9027 was serially exposed to sub-MIC level of ciprofloxacin for 14 days to generate E1 strain, then cultured in antibiotic-free environment for 10 days to obtain E2 strain. Changes in MIC values, susceptibility profile and virulence (protease, elastin, biofilm formation, motility) of these strains were evaluated compared to the original P. aeruginosa ATCC 9027. After antibiotic exposure, P. aeruginosa strains developed resistance to the ciprofloxacin with the MIC values increased by 32 times, and the exposed strains only developed resistance to ciprofloxacin and other fluoroquinolones but not to unrelated antibiotics. The resistant phenotype did not revert fully after 10 days in antibiotic-free environment. In addition, there was noticeable changes in the pathogen’s virulence factors during antibiotic exposure. In summary, sub-MICs exposure of P. aeruginosa provided positive selection for antibiotic resistant phenotype, which did not revert after 10 days in antibiotic- free environment. Exposure to sub-MIC level of antibiotics caused significant changes in virulence and antibiotic susceptibility of the pathogen.

Scanning electron microscope (SEM) is a popular tool used for observing bacteria surface and morphology. Using Pseudomonas aeruginosa as a model, this work aimed to show a SEM preparative procedure that is simple and economical but does not result in considerable data loss. This was accomplished via testing fixing ability of 10% formalin versus 2.5% glutaraldehyde, efficiency of air drying versus t-butyl alcohol drying method. Following that, polypropylene, dialysis tubing and agar were also assessed for their ability to serve as a supporting material for cell adhesion in preparing sample for SEM. Consequently, obtained data showed that the procedure using 24-hour 10% formalin fixation and t-butyl alcohol drying preserved well bacterial morphology. With this procedure, little cell or membrane damage was seen while extracellular structures were clearly observed. Furthermore, when this procedure was applied with different types of substrates including polypropylene, dialysis tubing, and agar, it showed that sample fixed on polypropylene maintained well extracellular structures meanwhile sample fixed on agar presented well bacterial morphology. In conclusion, our data suggested that coating samples on polypropylene, followed by 24-hour 10% formalin fixation and t-butyl alcohol drying was appropriate for observing bacteria under SEM.

Background: Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous bacterium that can be found in most moisture places, such as soil, water, food, plants, and animals, including humans. Due to genetic flexibility among strains, there is no standard molecular identification for P. aeruginosa from different sources. In this study, monoplex and duplex PCR targeting oprL, algD and nfxB were assessed for the identification of commensal P. aeruginosa. Methods: Twenty-nine commensal Pseudomonas isolates, including 16 P. aeruginosa isolates and 13 P. aeruginosa-like isolates, were used in the study. First, monoplex PCR targeting oprL, algD and nfxB using published primers was carried out to test their ability to detect commensal Pseudomonas isolates. Then, two new primer pairs targeting oprL were designed (oprL-pp1 and oprL-pp2) and used for oprL-algD duplex PCR to check for the improvement of commensal P. aeruginosa detection. Result and conclusion: AlgD or nfxB monoplex PCR had the same sensitivity of 93.75% and specificity of 100%, while oprL PCR using published primers was more sensitive (100%) but less specific (0%). Duplex PCR yielded high sensitivity and specificity in detecting P. aeruginosa. Both oprL-pp1/algD and oprL-pp2/algD duplex-PCR had 93.75% sensitivity (15/16 P. aeruginosa isolates) and 100% specificity (0/13 P. aeruginosa-like isolates). In addition, oprL-pp2 primers were more specific than oprL-pp1 primers, with only 2 of 13 P. aeruginosa-like isolates detected, while oprL-pp1 primers detected all P. aeruginosa-like isolates. Compared to the monoplex PCR that only targeted the oprL gene, the duplex PCR utilizing oprL-pp2 and algD primers identified 15/16 P. aeruginosa isolates (93.75% specificity). Additionally, the duplex PCR used in this study was negative for non-Pseudomonas species, including E. coli, V. cholera, V. parahaemolyticus, and S. aureus. In conclusion, our duplex PCR targeting oprL and algD could be a valuable tool for commensal P. aeruginosa screening.

Aim: Recently, the rise in Staphylococcal infection incidence accompanied by a rise of antibiotic-resistant strains is a major threat to public health. In this study, mechanisms leading to the occurrence of high-level multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains after fluoroquinolone (FQ) exposure were investigated. Methodology: Serially exposing S. aureus ATCC 29213 to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days was performed to obtain S. aureus -1 strains and culturing for another 10 days without antibiotics to obtain S. aureus-2 strains. The genomic alterations in FQ-exposed strains were reached using whole genome sequencing and target sequencing. The expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance were evaluated using RT-qPCR. Results: After serial FQ exposure, we observed a strong and irreversible increase of MICs to all applied FQs, i.e 32 to 128 times in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 significant mutations including 2 deletions, 1 insertion, and 7 missense mutations that occur in all S. aureus-1 and -2 but not in initial strain. The FQ target, GrlA, was also mutated (R570H) in all S. aureus-1 and -2 which can partly explain the development of FQ resistance over the FQ exposure. Besides, FQ exposure also resulted in overexpression of genes encoding for (1) efflux pumps and their regulator (norA, norB, norC, and mgrA); (2) alternative sigma factors (sigB and sigS); (3) acetyltransferase (rimI); (4) methicillin resistance (fmtB); and (5) hypothetical protein BJI72_0645. Conclusion: The mutations occurred in the FQ-target sequence were associated with high-level FQ resistance while the activation of efflux pump systems and post-translational modification proteins played an important role in the emergence of MDR in S. aureus.

Aim Fluoroquinolone (FQ) is a potent antibiotic class. However, resistance to this class emerges quickly which hinders its application. In this study, mechanisms leading to the emergence of multidrug-resistant (MDR) Staphylococcus aureus (S. aureus) strains under FQ exposure were investigated. Methodology S. aureus ATCC 29213 was serially exposed to ciprofloxacin (CIP), ofloxacin (OFL), or levofloxacin (LEV) at sub-minimum inhibitory concentrations (sub-MICs) for 12 days to obtain S. aureus -1 strains and antibiotic-free cultured for another 10 days to obtain S. aureus-2 strains. The whole genome (WGS) and target sequencing were applied to analyze genomic alterations; and RT-qPCR was used to access the expressions of efflux-related genes, alternative sigma factors, and genes involved in FQ resistance. Results A strong and irreversible increase of MICs was observed in all applied FQs (32 to 128 times) in all S. aureus-1 and remained 16 to 32 times in all S. aureus-2. WGS indicated 10 noticeable mutations occurring in all FQ-exposed S. aureus including 2 insdel mutations in SACOL0573 and rimI; a synonymous mutation in hslO; and 7 missense mutations located in an untranslated region. GrlA, was found mutated (R570H) in all S. aureus-1 and -2. Genes encoding for efflux pumps and their regulator (norA, norB, norC, and mgrA); alternative sigma factors (sigB and sigS); acetyltransferase (rimI); methicillin resistance (fmtB); and hypothetical protein BJI72_0645 were overexpressed in FQ-exposed strains. Conclusion The emergence of MDR S. aureus was associated with the mutations in the FQ-target sequences and the overexpression of efflux pump systems and their regulators.