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Antimicrobial susceptibility and virulence genes of clinical and environmental isolates of Pseudomonas aeruginosa.

ABSTRACT: Background:Pseudomonas aeruginosa is ubiquitous, has intrinsic antibiotic resistance mechanisms, and is associated with serious hospital-associated infections. It has evolved from being a burn wound infection into a major nosocomial threat. In this study, we compared and correlated the antimicrobial resistance, virulence traits and clonal relatedness between clinical and fresh water environmental isolates of P. aeruginosa. Methods:219 P. aeruginosa isolates were studied: (a) 105 clinical isolates from 1977 to 1985 (n = 52) and 2015 (n = 53), and (b) 114 environmental isolates from different fresh water sources. All isolates were subjected to ERIC-PCR typing, antimicrobial susceptibility testing and virulence factor genes screening. Results:Clinical and environmental isolates of P. aeruginosa were genetically heterogenous, with only four clinical isolates showing 100% identical ERIC-PCR patterns to seven environmental isolates. Most of the clinical and environmental isolates were sensitive to almost all of the antipseudomonal drugs, except for ticarcillin/clavulanic acid. Increased resistant isolates was seen in 2015 compared to that of the archived isolates; four MDR strains were detected and all were retrieved in 2015. All clinical isolates retrieved from 1977 to 1985 were susceptible to ceftazidime and ciprofloxacin; but in comparison, the clinical isolates recovered in 2015 exhibited 9.4% resistance to ceftazidime and 5.7% to ciprofloxacin; a rise in resistance to imipenem (3.8% to 7.5%), piperacillin (9.6% to 11.3%) and amikacin (1.9% to 5.7%) and a slight drop in resistance rates to piperacillin/tazobactam (7.7% to 7.5%), ticarcillin/clavulanic acid (19.2% to 18.9%), meropenem (15.4% to 7.5%), doripenem (11.5% to 7.5%), gentamicin (7.7% to 7.5%) and netilmicin (7.7% to 7.5%). Environmental isolates were resistant to piperacillin/tazobactam (1.8%), ciprofloxacin (1.8%), piperacillin (4.4%) and carbapenems (doripenem 11.4%, meropenem 8.8% and imipenem 2.6%). Both clinical and environmental isolates showed high prevalence of virulence factor genes, but none were detected in 10 (9.5%) clinical and 18 (15.8%) environmental isolates. The exoT gene was not detected in any of the clinical isolates. Resistance to carbapenems (meropenem, doripenem and imipenem), β-lactamase inhibitors (ticarcillin/clavulanic acid and piperacillin/tazobactam), piperacillin, ceftazidime and ciprofloxacin was observed in some of the isolates without virulence factor genes. Five virulence-negative isolates were susceptible to all of the antimicrobials. Only one MDR strain harbored none of the virulence factor genes. Conclusion:Over a period of 30 years, a rise in antipseudomonal drug resistance particularly to ceftazidime and ciprofloxacin was observed in two hospitals in Malaysia. The occurrence of resistant environmental isolates from densely populated areas is relevant and gives rise to collective anxiety to the community at large.

SUBMITTER: Liew SM

PROVIDER: S-EPMC6346980 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Quorum sensing (QS)-based transcriptional responses in Pseudomonas aeruginosa have been defined on the basis of increases in transcript levels of QS-controlled genes such as lasB and aprA following the hierarchical transcriptional increases of central controllers such as the lasR gene. These increases occur at high bacterial concentrations such as early-stationary-phase growth in vitro. However, the extent to which the increases occur in a variety of clinical and environmental isolates has not been determined nor is there extensive information on allelic variation in lasR genes. An analysis of the sequences of the lasR gene among 66 clinical and environmental isolates showed that 81% have a sequence either identical to that of strain PAO1 or with a silent mutation, 15% have nucleotide changes resulting in amino acid changes, and 5% have an insertion sequence in the lasR gene. Using real-time PCR to quantify transcript levels of lasR, lasB, and aprA in the early log and early stationary phases among 35 isolates from bacteremia and pneumonia cases and the environment, we found most (33 of 35) strains had increases in lasR transcripts in early stationary phase but with a very wide range of final transcript levels per cell. There was a strong correlation (r(2) = 0.84) between early-log- and early-stationary-phase transcript levels in all strains, but this finding remained true only for the 50% of strains above the median level of lasR found in early log phase. There were significant (P < 0.05) but weak-to-modest correlations of lasR transcript levels with aprA (r(2) = 0.2) and lasB (r(2) = 0.5) transcript levels, but again this correlation occurred only in the 50% of P. aeruginosa strains with the highest levels of lasR transcripts in early stationary phase. There were no differences in distribution of lasR alleles among the bacteremia, pneumonia, or environmental isolates. Overall, only about 50% of P. aeruginosa strains from clinical and environmental sources show a lasR-dependent increase in the transcription of aprA and lasB genes, indicating that for about 50% of clinical isolates this regulatory system may not play a significant role in pathogenesis.

Project description:BackgroundEmergence of multi-drug resistant Pseudomonas aeruginosa, coupled with the pathogen's versatile virulence factors, lead to high morbidity and mortality rates. The current study investigated the potential association between the antibiotic resistance and the production of virulence factors among P. aeruginosa clinical isolates collected from Alexandria Main University Hospital in Egypt. We also evaluated the potential of the phenotypic detection of virulence factors to reflect virulence as detected by virulence genes presence. The role of alginate in the formation of biofilms and the effect of ambroxol, a mucolytic agent, on the inhibition of biofilm formation were investigated.ResultsA multi-drug resistant phenotype was detected among 79.8% of the isolates. The most predominant virulence factor was biofilm formation (89.4%), while DNase was least detected (10.6%). Pigment production was significantly associated with ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to cefepime, and DNase production was significantly associated with intermediate resistance to meropenem. Among the tested virulence genes, lasB and algD showed the highest prevalence rates (93.3% and 91.3%, respectively), while toxA and plcN were the least detected ones (46.2% and 53.8%, respectively). Significant association of toxA with ceftazidime susceptibility, exoS with ceftazidime and aztreonam susceptibility, and plcH with piperacillin-tazobactam susceptibility was observed. There was a significant correlation between alkaline protease production and the detection of algD, lasB, exoS, plcH and plcN; pigment production and the presence of algD, lasB, toxA and exoS; and gelatinase production and the existence of lasB, exoS and plcH. Ambroxol showed a high anti-biofilm activity (5% to 92%). Quantitative reverse transcriptase polymerase chain reaction showed that alginate was not an essential matrix component in P. aeruginosa biofilms.ConclusionsHigh virulence coupled with the isolates' multi-drug resistance to commonly used antimicrobials would increase morbidity and mortality rates among P. aeruginosa infections. Ambroxol that displayed anti-biofilm action could be suggested as an alternative treatment option, yet in vivo studies are required to confirm these findings. We recommend active surveillance of antimicrobial resistance and virulence determinant prevalence for better understanding of coregulatory mechanisms.

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Antimicrobial susceptibility and virulence genes of clinical and environmental isolates of Pseudomonas aeruginosa.

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