Objectives To investigate the potential relationship between quinolone resistance and biofilm production in a collection of Salmonella enterica clinical isolates and in S. enterica serovar Typhimurium serial mutants with increasing resistance to ciprofloxacin. Methods Nalidixic acid susceptibility and biofilm formation were assessed in a collection of 122 S. enterica clinical isolates. An in vitro quinolone-resistant mutant, 59-64, was obtained from a biofilm-producing and quinolone-susceptible clinical isolate, 59-wt, in a multistep selection process after increasing ciprofloxacin concentrations. The quinolone resistance mechanisms [target gene and multidrug resistance (MDR) regulatory mutations, MICs of several antibiotics, cell envelope protein analysis, real-time PCR and ciprofloxacin accumulation] were characterized for mutant strains. In addition, analysis of fitness, biofilm formation, rdar morphotype and expression of biofilm-related genes by real-time PCR were also determined. Results Nalidixic acid-susceptible S. enterica strains were more prevalent in producing biofilm than the resistant counterparts. Strain 59-64 acquired five target gene mutations and showed an MDR phenotype. AcrAB and acrF overexpression were ruled out, whereas TolC did show increased expression in 59-64, which, in addition, accumulated less ciprofloxacin. Consistently, increased ramA expression was seen in 59-64 and attributed to a mutation within its promoter. Reduced biofilm production related to diminished csgB expression as well as reduced fitness was seen for 59-64, which was unable to form the rdar morphotype. Conclusions Quinolone resistance acquisition may be associated with decreased production of biofilm due to lower csgB expression. Efflux, biofilm production and fitness seem to be interrelated.

The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents. PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT–PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing. We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam. Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.

Infectious diseases are emerging and re-emerging due to climate change. Understanding how climate variability affects the transmission of infectious diseases is important for both researchers and the general public. Yet, the widespread knowledge of the general public on this matter is unknown, and quantitative research is still lacking. A survey was designed to assess the knowledge and perception of 1) infectious diseases, 2) climate change and 3) the effect of climate change on infectious diseases. Participants were recruited via convenience sampling, and an anonymous cross-sectional survey with informed consent was distributed to each participant. Descriptive and inferential analyses were performed primarily focusing on the occupational background as well as nationality of participants. A total of 458 individuals participated in this study, and most participants were originally from Myanmar, the Netherlands, Spain, United Kingdom and the United States. Almost half (44%) had a background in natural sciences and had a higher level of knowledge on infectious diseases compared to participants with non-science background (mean score of 12.5 and 11.2 out of 20, respectively). The knowledge of the effect of climate change on infectious diseases was also significantly different between participants with and without a background in natural sciences (13.1 and 11.8 out of 20, respectively). The level of knowledge on various topics was highly correlated with nationality but not associated with age. The general population demonstrated a high awareness and strong knowledge of climate change regardless of their background in natural sciences. This study exposes a knowledge gap in the general public regarding the effect of climate change on infectious diseases, and highlights that different levels of knowledge are observed in groups with differing occupations and nationalities. These results may help to develop awareness interventions for the general public.

This study was focused on analysing the heterogeneity of mutations occurring in the regulators of efflux-mediated MDR in Salmonella Typhimurium. Moreover, the impact of such mutations on impairing the transcription of ramA, acrB, tolC and acrF was also assessed as was the impact on the resistance or decreased susceptibility phenotype. Strains were selected in vitro under increasing ciprofloxacin concentrations. Etest and broth microdilution tests were used to determine the MICs of several unrelated compounds. Screening of mutations in the quinolone target genes and MDR regulators was performed. RT–PCR analysis was used to detect the levels of expression of acrB, tolC, ompF, acrF, emrB, acrR, ramA, soxS and marA. All mutant strains showed increased MICs of most of the antimicrobials tested, with the exception of kanamycin. Mutations in the quinolone target genes did not occur in all the mutants, which all harboured mutations in the ramRA regulatory region. All the mutants overexpressed ramA, tolC and acrB (only tested in 60-wt derivatives), whereas differential results were seen for the remaining genes. Mutations in the ramRA region related to resistance and/or decreased susceptibility to antimicrobials predominate in Salmonella. There is heterogeneity in the types of mutations, with deletions affecting RamR-binding sites having a greater impact on ramA expression and the MDR phenotype.