Background: Stenotrophomonas maltophilia is a nosocomial opportunistic pathogen of the Xanthomonadaceae. The organism has been isolated from both clinical and soil environments in addition to the sputum of cystic fibrosis patients and the immunocompromised. Whilst relatively distant phylogenetically, the closest sequenced relatives of S. maltophilia are the plant pathogenic xanthomonads.

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.Pseudomonas aeruginosa is an opportunistic human pathogen that demonstrates an innate resistance to multiple classes of antimicrobials (5), a property explained in part by the operation of multidrug efflux systems of the resistance-nodulation division (RND) family (19,20). Several RND-type multidrug efflux systems have been described in P. aeruginosa (20), although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (4, 10, 21). MexAB-OprM exports a variety of clinically used antimicrobials (including several classes of antibiotics and biocides) (19,20) but, as well, several additional agents with antimicrobial activity (e.g., dyes [9], detergents [34], and organic solvents such as aromatic hydrocarbons [11]) and acylhomoserine lactones (AHLs) associated with quorum sensing (3, 18). AHLs play a role in cell density-dependent expression of a number of virulence factors in P. aeruginosa and, thus, the activity of this efflux system can influence virulence (6). The observation that MexAB-OprM overproduction can compromise bacterial "fitness" (29) and that pump-overproducing mutants of P. aeruginosa can be selected in animal models of infection in the absence of antimicrobial selection (7) also point to in vivo functions for this pump independent of antimicrobial efflux and resistance.Hyperproduction of MexAB-OprM has been documented in so-called nalB mutants (13) carrying lesions in the mexR gene (13,26,35,38) encoding a repressor of mexAB-oprM expression (22,35). MexR is a member of the MarR family of regulators (16) and binds as a dimer (12) to two sites in the mexRmexA intragenic region, near mexR and overlapping promoters for mexR and mexAB-oprM (2). MexAB-OprM hyperexpression also occurs independently of mutations in mexR; these so-called nalC mutants (1, 13, 35) carry a mutation in a gene (PA3721, also known as nalC) that encodes a TetR family repressor of an adjacent two-gene operon, PA3720-PA3719 (1). It is, in fact, the increased expression of PA3719 that results from disruption of the nalC repressor gene that promotes mexAB-oprM hyperexpression (1), apparently as a result of PA3719 modulation of MexR repressor activity (L. Cao, S.

Objectives Third-generation cephalosporin-resistant Escherichia coli from community-acquired urinary tract infections are increasingly reported worldwide. We sought to determine and characterize the mechanisms of cefotaxime resistance employed by urinary E. coli obtained from primary care, over 12 months, in Bristol and surrounding counties in South-West England. Methods Cefalexin-resistant E. coli isolates were identified from GP-referred urine samples using disc susceptibility testing. Cefotaxime resistance was determined by subsequent plating onto MIC breakpoint plates. β-Lactamase genes were detected by PCR. WGS was performed on 225 isolates and analyses were performed using the Center for Genomic Epidemiology platform. Patient information provided by the referring general practices was reviewed. Results Cefalexin-resistant E. coli (n=900) isolates were obtained from urines from 146 general practices. Following deduplication by patient approximately 69% (576/836) of isolates were cefotaxime resistant. WGS of 225 isolates identified that the most common cefotaxime-resistance mechanism was blaCTX-M carriage (185/225), followed by plasmid-mediated AmpCs (pAmpCs) (17/225), AmpC hyperproduction (13/225), ESBL blaSHV variants (6/225) or a combination of both blaCTX-M and pAmpC (4/225). Forty-four STs were identified, with ST131 representing 101/225 isolates, within which clade C2 was dominant (54/101). Ciprofloxacin resistance was observed in 128/225 (56.9%) of sequenced isolates, predominantly associated with fluoroquinolone-resistant clones ST131 and ST1193. Conclusions Most cefalexin-resistant E. coli isolates were cefotaxime resistant, predominantly caused by blaCTX-M carriage. The correlation between cefotaxime resistance and ciprofloxacin resistance was largely attributable to the high-risk pandemic clones ST131 and ST1193. Localized epidemiological data provide greater resolution than regional data and can be valuable for informing treatment choices in the primary care setting.

Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed animal populations. We conducted whole genome sequencing analysis of 138 representative 3GC-R isolates previously collected from dairy farms in South West England and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates: encoding CTX-M-1, -14, -15, -32 and the novel variant, CTX-M-214), blaCMY-2 (6 isolates) and blaDHA-1 (one isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ∼220 kb IncHI2 plasmid carrying blaCTX-M-32 was sequenced to closure and designated pMOO-32. It was found experimentally to be stable in cattle and human transconjugant E. coli even in the absence of selective pressure and was found by multiplex PCR to be present on 26 study farms representing a remarkable range of transmission over 1500 square kilometres. However, the plasmid was not found amongst human urinary E. coli we have recently characterised from people living in the same geographical location, collected in parallel with farm sampling. There were close relatives of two blaCTX-M plasmids circulating amongst eight human and two cattle isolates, and a closely related blaCMY-2 plasmid found in one cattle and one human isolate. However, phylogenetic evidence of recent sharing of 3GC-R strains between farms and humans in the same region was not found. Importance Third-generation cephalosporins (3GCs) are critically important antibacterials and 3GC-resistance (3GC-R) threatens human health, particularly in the context of opportunistic pathogens such as Escherichia coli. There is some evidence for zoonotic transmission of 3GC-R E. coli through food, but little work has been done examining possible transmission (e.g. via interaction of people with the local near-farm environment). We characterised acquired 3GC-R E. coli found on dairy farms in a geographically restricted region of the United Kingdom and compared these with E. coli from people living in the same region, collected in parallel. Whilst there is strong evidence for recent farm-to-farm transmission of 3GC-R strains and plasmids – including one epidemic plasmid that has a remarkable capacity to transmit – there was no evidence that 3GC-R found on study farms had a significant impact on circulating 3GC-R E. coli strains or plasmids in the local human population.

The majority of S. maltophilia clinical isolates behave similarly in terms of beta-lactamase expression and beta-lactam resistance properties, despite considerable phylogenetic variability.