Growth in a biofilm promotes conjugation of a bla NDM-1-bearing plasmid between Klebsiella pneumoniae strains.

Antimicrobial resistance (AMR) is a growing problem, especially in Gram-negative Enterobacteriaceae such as Klebsiella pneumoniae. Horizontal transfer of conjugative plasmids contributes to AMR gene dissemination. Bacteria such as K. pneumoniae commonly exist in biofilms, yet most studies focus on planktonic cultures. Here we studied the transfer of a multi-drug resistance plasmid in planktonic and biofilm populations of K. pneumoniae. We determined plasmid transfer from a clinical isolate, CPE16, which carried four plasmids, including the 119-kbp blaNDM-1-bearing F-type plasmid pCPE16_3, in planktonic and biofilm conditions. We found that transfer frequency of pCPE16_3 in a biofilm was orders-of-magnitude higher than between planktonic cells. In 5/7 sequenced transconjugants (TCs) multiple plasmids had transferred. Plasmid acquisition had no detectable growth impact on TCs. Gene expression of the recipient and a transconjugant was investigated by RNA-sequencing in three lifestyles: planktonic exponential growth, planktonic stationary phase, and biofilm. We found that lifestyle had a substantial impact on chromosomal gene expression, and plasmid carriage affected chromosomal gene expression most in stationary planktonic and biofilm lifestyles. Furthermore, expression of plasmid genes was lifestyle-dependent, with distinct signatures across the three conditions. Our study shows that growth in biofilm greatly increased the risk of conjugative transfer of a carbapenem resistance plasmid in K. pneumoniae without fitness costs and minimal transcriptional rearrangements, thus highlighting the importance of biofilms in the spread of AMR in this opportunistic pathogen. IMPORTANCE Carbapenem-resistant K. pneumoniae is particularly problematic in hospital settings. Carbapenem resistance genes can transfer between bacteria via plasmid conjugation. Alongside drug resistance, K. pneumoniae can form biofilms on hospital surfaces, at infection sites and on implanted devices. Biofilms are naturally protected and can be inherently more tolerant to antimicrobials than their free-floating counterparts. There have been indications that plasmid transfer may be more likely in biofilm populations, thus creating a conjugation "hotspot". However, there is no clear consensus on the effect of the biofilm lifestyle on plasmid transfer. Therefore, we aimed to explore the transfer of a plasmid in planktonic and biofilm conditions, and the impact of plasmid acquisition on a new bacterial host. Our data show transfer of a resistance plasmid is increased in a biofilm, which may be a significant contributing factor to the rapid dissemination of resistance plasmids in K. pneumoniae.

Antimicrobial resistance and gene regulation in Enteroaggregative Escherichia coli from Egyptian children with diarrhoea: Similarities and differences.

Enteroaggregative Escherichia coli (EAEC) is a common diarrhoeagenic human pathogen, isolated from patients in both developing and industrialized countries, that is becoming increasingly resistant to many frontline antibiotics. In this study, we screened 50 E. coli strains from children presenting with diarrhea at the outpatients clinic of Assiut University Children's Hospital, Egypt. We show that all of these isolates were resistant to multiple classes of antibiotics and identified two as being typical EAEC strains. Using whole genome sequencing, we determined that both isolates carried, amongst others, blaCTX-M and blaTEM antibiotic resistance genes, as well as many classical EAEC virulence determinants, including the transcriptional regulator, AggR. We demonstrate that the expression of these virulence determinants is dependent on AggR, including aar, which encodes for a repressor of AggR, Aar. Since biofilm formation is the hallmark of EAEC infection, we examined the effect of Aar overexpression on both biofilm formation and AggR-dependent gene expression. We show that whilst Aar has a minimal effect on AggR-dependent transcription it is able to completely disrupt biofilm formation, suggesting that Aar affects these two processes differently. Taken together, our results suggest a model for the induction of virulence gene expression in EAEC that may explain the ubiquity of EAEC in both sick and healthy individuals.

New Multidrug Efflux Inhibitors for Gram-Negative Bacteria.

Active efflux of antibiotics preventing their accumulation to toxic intracellular concentrations contributes to clinically relevant multidrug resistance. Inhibition of active efflux potentiates antibiotic activity, indicating that efflux inhibitors could be used in combination with antibiotics to reverse drug resistance. Expression of ramA by Salmonella enterica serovar Typhimurium increases in response to efflux inhibition, irrespective of the mode of inhibition. We hypothesized that measuring ramA promoter activity could act as a reporter of efflux inhibition. A rapid, inexpensive, and high-throughput green fluorescent protein (GFP) screen to identify efflux inhibitors was developed, validated, and implemented. Two chemical compound libraries were screened for compounds that increased GFP production. Fifty of the compounds in the 1,200-compound Prestwick chemical library were identified as potential efflux inhibitors, including the previously characterized efflux inhibitors mefloquine and thioridazine. There were 107 hits from a library of 47,168 proprietary compounds from L. Hoffmann La Roche; 45 were confirmed hits, and a dose response was determined. Dye efflux and accumulation assays showed that 40 Roche and three Prestwick chemical library compounds were efflux inhibitors. Most compounds had specific efflux-inhibitor-antibiotic combinations and/or species-specific synergy in antibiotic disc diffusion and checkerboard assays performed with Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and Salmonella Typhimurium. These data indicate that both narrow-spectrum and broad-spectrum combinations of efflux inhibitors with antibiotics can be found. Eleven novel efflux inhibitor compounds potentiated antibiotic activities against at least one species of Gram-negative bacteria, and data revealing an E. coli mutant with loss of AcrB function suggested that these are AcrB inhibitors.IMPORTANCE Multidrug-resistant Gram-negative bacteria pose a serious threat to human and animal health. Molecules that inhibit multidrug efflux offer an alternative approach to resolving the challenges caused by antibiotic resistance, by potentiating the activity of old, licensed, and new antibiotics. We have developed, validated, and implemented a high-throughput screen and used it to identify efflux inhibitors from two compound libraries selected for their high chemical and pharmacological diversity. We found that the new high-throughput screen is a valuable tool to identify efflux inhibitors, as evidenced by the 43 new efflux inhibitors described in this study.