Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress.

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance.

Implications of two-component systems EnvZ/OmpR and BaeS/BaeR in in vitro temocillin resistance in Escherichia coli.

BACKGROUND: BaeS/BaeR is a two-component system of Escherichia coli that controls the expression of porins and efflux pumps. Its role in beta-lactam resistance is limited. OBJECTIVES: To study the role of baeS/baeR two-component system in temocillin resistance in E. coli. METHODS: E. coli strain BW25113 and single-gene deletion mutants related to two-component systems were collected from the KEIO collection. Double-gen deletion mutants were generated. Temocillin-resistant mutant frequencies were determined at 32 mg/L. E. coli BW25113 mutants were selected by selective pressure from serial passages. Biological costs were analysed by growth curves. Genomes of the generated mutants were sequenced. The expression level of the mdtA, mdtB, mdtC, acrD and tolC in the ΔbaeS mutant was determined by RT-PCR (with/without temocillin exposure). RESULTS: The frequency of temocillin mutants ranged from 2.12 × 10-8 to 4.51 × 10-8 in single-porin mutants. No mutants were recovered from E. coli BW25113 (>10-9). Selection of temocillin-resistant variants by serial passage yielded mutants up to 128 mg/L. Mutations were found in the baeS gene. Temocillin MICs ranged from 4 to 32 mg/L (highest MICs for ΔbaeS and ΔompR). The efflux pumps mdtA, mdtB, mdtC and acrD pumps were overexpressed 3-10-fold in the presence of temocillin in ΔbaeS compared to control. CONCLUSIONS: Mutations in the sensor histidine kinase, baeS, may be involved in temocillin resistance through the expression of the efflux pumps mdtABC and acrD. In addition, the low mutation rate may be a good predictor of temocillin activity.

Evaluation of temocillin efficacy against KPC-2-producing Klebsiella pneumoniae isolates in a hollow-fibre infection model.

BACKGROUND: Temocillin is an old antimicrobial that is resistant to hydrolysis by ESBLs but has variable activity against carbapenemase-producing Enterobacteriaceae. The current EUCAST susceptibility breakpoints for Enterobacterales are set at ≤16 mg/L (susceptible with increased exposure) based on a dose of 2 g q8h, but there is limited information on the efficacy of this dose against temocillin-susceptible carbapenemase-producing Klebsiella pneumoniae isolates. OBJECTIVES: To evaluate the efficacy of this dose using a hollow-fibre infection model (HFIM) against six KPC-2-producing clinical isolates of K. pneumoniae. METHODS: The isolates were characterized by WGS and temocillin susceptibility was determined using standard and high inoculum temocillin. Mutant frequencies were estimated and temocillin activity was tested in time-kill assays and in the HFIM. At standard conditions, three of the isolates were classified as susceptible (MIC ≤ 16 mg/L) and three as resistant (MIC > 16 mg/L). The HFIM was performed over 3 days to mimic human-like pharmacokinetics of 2 g q8h. Bacterial counts were performed by plating on Mueller-Hinton agar (MHA) and MHA containing 64 mg/L temocillin to detect resistant subpopulations. RESULTS: All isolates showed a reduction in bacterial population of at least 3 log cfu/mL within the first 8 h of simulated treatment in the hollow-fibre assay. Regrowth was observed for the three resistant isolates and one of the susceptible ones. The MIC value for these isolates was higher by at least two dilutions compared with their initial values. CONCLUSIONS: These data suggest that an optimized pharmacokinetic regimen may be of clinical interest for the treatment of KPC-2-producing K. pneumoniae susceptible to temocillin. These data showed activity of temocillin against KPC-2-producing K. pneumoniae susceptible to temocillin; however, a dose of 2g q8h administered over 30 min may be inadequate to prevent the emergence of resistant variants.

The mef(A)/msr(D)-carrying streptococcal prophage Φ1207.3 encodes an SOS-like system, induced by UV-C light, responsible for increased survival and increased mutation rate.

Bacterial SOS response is an inducible system of DNA repair and mutagenesis. Streptococci lack a canonical SOS response, but an SOS-like response was reported in some species. The mef(A)-msr(D)-carrying prophage Ф1207.3 of Streptococcus pyogenes contains a region, spanning orf6 to orf11, showing homology to characterized streptococcal SOS-like cassettes. Genome-wide homology search showed the presence of the whole Φ1207.3 SOS-like cassette in three S. pyogenes prophages, while parts of it were found in other bacterial species. To investigate whether this cassette confers an SOS-mutagenesis phenotype, we constructed Streptococcus pneumoniae R6 isogenic derivative strains: (i) FR172, streptomycin resistant, (ii) FR173, carrying Φ1207.3, and (iii) FR174, carrying a recombinant Φ1207.3, where the SOS-like cassette was deleted. These strains were used in survival and mutation rate assays using a UV-C LED instrument, for which we designed and 3D-printed a customized equipment, constituted of an instrument support and swappable-autoclavable mini-plates and lids. Upon exposure to UV fluences ranging from 0 to 6,400 J/m2 at four different wavelengths, 255, 265, 275, and 285 nm, we found that the presence of Φ1207.3 SOS-like cassette increases bacterial survival up to 34-fold. Mutation rate was determined by measuring rifampicin resistance acquisition upon exposure to UV fluence of 50 J/m2 at the four wavelengths by fluctuation test. The presence of Φ1207.3 SOS-like cassette resulted in a significant increase in the mutation rate (up to 18-fold) at every wavelength. In conclusion, we demonstrated that Φ1207.3 carries a functional SOS-like cassette responsible for an increased survival and increased mutation rate in S. pneumoniae. IMPORTANCE Bacterial mutation rate is generally low, but stress conditions and DNA damage can induce stress response systems, which allow for improved survival and continuous replication. The SOS response is a DNA repair mechanism activated by some bacteria in response to stressful conditions, which leads to a temporary hypermutable phenotype and is usually absent in streptococcal genomes. Here, using a reproducible and controlled UV irradiation system, we demonstrated that the SOS-like gene cassette of prophage Φ1207.3 is functional, responsible for a temporary hypermutable phenotype, and enhances bacterial survival to UV irradiation. Prophage Φ1207.3 also carries erythromycin resistance genes and can lysogenize different pathogenic bacteria, constituting an example of a mobile genetic element which can confer multiple phenotypes to its host.

Higher prevalence of CTX-M-27-producing Escherichia coli belonging to ST131 clade C1 among residents of two long-term care facilities in Southern Spain.

Recently, the emergence of an international lineage of the CTX-M-27-producing clade C1 of Escherichia coli ST131 is being observed. The aim is to see if this strain has also been introduced in our area. Twenty-eight (33%) out of 86 individuals from two LTCFs in Seville were found to be colonized with fluoroquinolone-resistant E. coli ST131 and 46% isolates were ESBL/pAmpC producers. C1 isolates were more common than C2 and more frequently produced blaESBL/pAmpC genes (53% vs 33%). Strain sharing was observed in 6 groups of 2-5 cases (61%). A differentiated cluster of 5 C1-CTX-M-27 isolates was found which lacked the M27PP1 region.

Interplay between IncF plasmids and topoisomerase mutations conferring quinolone resistance in the Escherichia coli ST131 clone: stability and resistance evolution.

The Escherichia coli ST131 H30-Rx subclone vehicles CTX-M-15 plasmids and mutations in gyrA and parC conferring multidrug resistance successfully in the clinical setting. The aim of this study was (1) to investigate the relationship of specific topoisomerase mutations on the stability of IncF (CTX-M producing) plasmids using isogenic E. coli mutants and (2) to investigate the impact of the IncF-type plasmids present in the E. coli clone ST131 on the evolution of quinolone resistance. E. coli ATCC 25922 (background strain) and derived mutants encoding specific QRDR substitutions were used. Also, NGS-characterized IncFIA and IncFIB plasmids (encoding CTX-M genes) were included. Plasmid stability was evaluated by sequential dilutions into Luria broth medium without antibiotics for 7 days. Mutant frequency to ciprofloxacin was also evaluated. Moderate differences in the IncF plasmids stability were observed among E. coli ATCC 25922 and isogenic mutants. Under our experimental conditions, the fluctuation of bacteria harboring plasmids was less than 0.5-log(10) in all cases. In the mutant frequency tests, it was observed that the presence of these IncF plasmids increased this value significantly (10-1000-fold). Quinolone resistance substitutions in gyrA or parC genes, frequently found associated with E. coli clone ST131, do not modify the stability of ST131-associated IncFIA and IncFIB plasmids under in vitro conditions. IncF-type plasmids present in E. coli clone ST131 facilitate the selection of resistance to quinolones. These results are consistent with the clinical scenario in which the combination of resistance to quinolones and beta-lactams is highly frequent in the E. coli clone ST131.

Contribution of hypermutation to fosfomycin heteroresistance in Escherichia coli.

OBJECTIVES: To explore the effect of combining defects in DNA repair systems with the presence of fosfomycin-resistant mechanisms to explain the mechanisms underlying fosfomycin heteroresistance phenotypes in Enterobacteriaceae. MATERIALS AND METHODS: We used 11 clinical Escherichia coli isolates together with isogenic single-gene deletion mutants in the E. coli DNA repair system or associated with fosfomycin resistance, combined with double-gene deletion mutants. Fosfomycin MICs were determined by gradient strip assay (GSA) and broth microdilution (BMD). Mutant frequencies for rifampicin (100 mg/L) and fosfomycin (50 and 200 mg/L) were determined. Using two starting inocula, in vitro fosfomycin activity was assessed over 24 h in growth (0.5-512 mg/L) and time-kill assays (64 and 307 mg/L). RESULTS: Strong and weak mutator clinical isolates and single-gene deletion mutants, except for ΔuhpT and ΔdnaQ, were susceptible by GSA. By BMD, the percentage of resistant clinical isolates reached 36%. Single-gene deletion mutants showed BMD MICs similar to those for subpopulations by GSA. Strong mutators showed a higher probability of selecting fosfomycin mutants at higher concentrations. By combining the two mechanisms of mutation, MICs and ranges of resistant subpopulations increased, enabling strains to survive at higher fosfomycin concentrations in growth monitoring assays. In time-kill assays, high inocula increased survival by 37.5% at 64 mg/L fosfomycin, compared with low starting inocula. CONCLUSIONS: The origin and variability of the fosfomycin heteroresistance phenotype can be partially explained by high mutation frequencies together with mechanisms of fosfomycin resistance. Subpopulations should be considered until clinical meaning is established.

Plasmidic qnr Genes Confer Clinical Resistance to Ciprofloxacin under Urinary Tract Physiological Conditions.

Escherichia coli variants expressing plasmid-mediated qnr genes are usually susceptible to fluoroquinolones by standard susceptibility testing. Here we show that, under specific urinary tract physiological conditions, susceptible laboratory and clinical strains harboring qnr determinants become fully resistant to ciprofloxacin (CIP). Therefore, physiological conditions, mainly urine pH values, should be considered when performing susceptibility testing of CIP activity against E. coli in treating urinary tract infection (UTI) and for selecting appropriate antibiotics for UTI treatment.

Rapid detection of the plasmid-mediated quinolone resistance determinant AAC(6')-Ib-cr in Enterobacteriaceae by MALDI-TOF MS analysis.

Objectives: Rapid detection of the plasmid-mediated quinolone resistance determinant AAC(6')-Ib-cr in Enterobacteriaceae by measuring acetyltransferase activity against fluoroquinolones by MALDI-TOF MS analysis. Methods: The presence of the AAC(6')-Ib-cr enzyme was determined by MS by measuring the acetyltransferase activity of a collection of 81 isogenic Escherichia coli control strains [10 carrying the AAC(6')-Ib-cr enzyme during exposure to ciprofloxacin, norfloxacin and levofloxacin] and further analysis of 36 clinical isolates [25 carrying the AAC(6')-Ib-cr enzyme in addition to different combinations of quinolone resistance mechanisms]. The effect of acetylation yields an increase of 43 Da in the mass of ciprofloxacin and norfloxacin, but not of levofloxacin, that can be observed by visual inspection of the mass peaks in the spectra. Results: Based on the characteristic peak pattern for the acetylated and non-acetylated forms of ciprofloxacin and norfloxacin, a clear differentiation between AAC(6')-Ib-cr-producing isolates and non-AAC(6')-Ib-cr-producing isolates was detected after an incubation time of 30 min, both in the isogenic control strains and in the clinical isolates. Levofloxacin was found intact. A 100% agreement was found between the MALDI-TOF-MS-based assay and the results of the molecular characterization of the tested isolates. Conclusions: MALDI-TOF MS is an outstanding method for detection of the AAC(6')-Ib-cr enzyme in clinical samples. The method is easy to perform and not time consuming, as analytical results can be obtained within minutes. For the first time, MALDI-TOF MS has been used to detect resistance promoted by enzymatic modification of antibiotics aside from ß-lactamases, expanding the capacity of analysis into new families of antibiotics.

Plasmid-mediated quinolone resistance: Two decades on.

After two decades of the discovery of plasmid-mediated quinolone resistance (PMQR), three different mechanisms have been associated to this phenomenon: target protection (Qnr proteins, including several families with multiple alleles), active efflux pumps (mainly QepA and OqxAB pumps) and drug modification [AAC(6')-Ib-cr acetyltransferase]. PMQR genes are usually associated with mobile or transposable elements on plasmids, and, in the case of qnr genes, are often incorporated into sul1-type integrons. PMQR has been found in clinical and environmental isolates around the world and appears to be spreading. Although the three PMQR mechanisms alone cause only low-level resistance to quinolones, they can complement other mechanisms of chromosomal resistance to reach clinical resistance level and facilitate the selection of higher-level resistance, raising a threat to the treatment of infections by microorganisms that host these mechanisms.

Urinary Tract Physiological Conditions Promote Ciprofloxacin Resistance in Low-Level-Quinolone-Resistant Escherichia coli.

Escherichia coli isolates carrying chromosomally encoded low-level-quinolone-resistant (LLQR) determinants are frequently found in urinary tract infections (UTIs). LLQR mutations are considered the first step in the evolutionary pathway producing high-level fluoroquinolone resistance. Therefore, their evolution and dissemination might influence the outcome of fluoroquinolone treatments of UTI. Previous studies support the notion that low urine pH decreases susceptibility to ciprofloxacin (CIP) in E. coli However, the effect of the urinary tract physiological parameters on the activity of ciprofloxacin against LLQR E. coli strains has received little attention. We have studied the activity of ciprofloxacin under physiological urinary tract conditions against a set of well-characterized isogenic E. coli derivatives carrying the most prevalent chromosomal mutations (ΔmarR, gyrA-S83L, gyrA-D87N, and parC-S80R and some combinations). The results presented here demonstrate that all the LLQR strains studied became resistant to ciprofloxacin (according to CLSI guidelines) under physiological conditions whereas the control strain lacking LLQR mutations did not. Moreover, the survival of some LLQR E. coli variants increased up to 100-fold after challenge with a high concentration of ciprofloxacin under UTI conditions compared to the results seen with Mueller-Hinton broth. These selective conditions could explain the high prevalence of LLQR mutations in E. coli Furthermore, our data strongly suggest that recommended methods for MIC determination produce poor estimations of CIP activity against LLQR E. coli in UTIs.

Impact of AAC(6')-Ib-cr in combination with chromosomal-mediated mechanisms on clinical quinolone resistance in Escherichia coli.

OBJECTIVES: aac(6')-Ib-cr is the most prevalent plasmid-mediated fluoroquinolone (FQ) resistance mechanism in Enterobacteriaceae. We aimed to analyse the interplay between this plasmid-mediated gene and chromosomal-mediated quinolone resistance mechanisms on both FQ resistance and bacterial fitness in Escherichia coli. METHODS: E. coli ATCC 25922 and derived isogenic strains carrying chromosomal-mediated quinolone resistance modifications (Ser83Leu-Asp87Asn in GyrA, Ser80Arg in ParC and/or a marR gene deletion) were electroporated with a pBK-CMV vector encoding AAC(6')-Ib-cr. The MICs of FQs were determined by microdilution and bactericidal activity was determined using time-kill curves. A peritoneal sepsis murine model was used to evaluate the in vivo impact. Bacterial fitness was analysed using growth curves and competition assays. RESULTS: The presence of the aac(6')-Ib-cr gene increased the MICs of ciprofloxacin and norfloxacin 4-8-fold for all E. coli genotypes, independently of the initial resistance level. Combination of the aac(6')-Ib-cr gene with three or four chromosomal mechanisms was necessary to reach MIC values above the susceptible category. Killing curve assays showed a clear selective advantage for survival in strains harbouring the aac(6')-Ib-cr gene (up to 7 log10 cfu/mL after 24 h). AAC(6')-Ib-cr significantly reduced the ciprofloxacin efficacy in vivo. In terms of bacterial fitness cost, maximal OD was significantly lower for all strains harbouring the aac(6')-Ib-cr gene, independently of chromosomal mutations associated. CONCLUSIONS: The aac(6')-Ib-cr gene, in spite of producing low-level resistance by itself, plays a relevant role in acquisition of a clinical level of ciprofloxacin and norfloxacin resistance, when combined with three or four chromosomal mutations, both in vitro and in vivo.

Comparison of clinical categories for Escherichia coli harboring specific qnr and chromosomal-mediated fluoroquinolone resistance determinants according to CLSI and EUCAST.

EUCAST breakpoints are more restrictive than those defined by CLSI. This study highlights the discrepancies between CLSI and EUCAST in a well characterized isogenic Escherichia coli collection and their correlations with specific quinolone resistance mechanisms. The greatest number of discrepancies was observed in strains containing 2-4 resistance mechanisms (MIC values on the borderline of clinical resistance). Bearing in mind that quinolones are concentration dependent antimicrobial agents, small changes in MIC may have relevant consequences for treatment outcomes.

Effect of the efflux pump QepA2 combined with chromosomally mediated mechanisms on quinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of the study was to determine the interplay between the plasmid-mediated qepA2 gene and multiple chromosomally mediated fluoroquinolone resistance determinants in the development of fluoroquinolone resistance in Escherichia coli and its influence on bacterial fitness. METHODS: E. coli ATCC 25922 and derived isogenic strains harbouring different chromosomally mediated fluoroquinolone resistance determinants were electroporated with pBK-CMV vector encoding QepA2. The MICs of fluoroquinolones were determined by standardized microdilution. The mutant prevention concentration (MPC) was evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays. RESULTS: The ciprofloxacin MIC for strains harbouring the qepA2 gene was 4- to 8-fold higher compared with strains without the qepA2 gene. The qepA2 gene also increased the MPC of ciprofloxacin 4- to 16-fold. Combination of the qepA2 gene plus two to three additional mechanisms conferred a clinically relevant resistance level. The presence of the qepA2 gene was associated with fitness costs in strains with mutations in the gyrA and/or parC genes, although the presence of an additional deletion of the marR gene compensated for this fitness cost by increasing bacterial fitness by 5%-23%. CONCLUSIONS: The additive effect of chromosomally mediated fluoroquinolone resistance mechanisms and the qepA2 gene led to clinical levels of fluoroquinolone resistance. Under competitive conditions, the qepA2 gene had a biological cost in E. coli that was compensated for by the presence of an additional deletion in the marR gene.

Challenges to accurate susceptibility testing and interpretation of quinolone resistance in Enterobacteriaceae: results of a Spanish multicentre study.

OBJECTIVES: The objective of this study was to evaluate the proficiency of Spanish laboratories with respect to accurate susceptibility testing and the detection and interpretation of quinolone resistance phenotypes in Enterobacteriaceae. METHODS: Thirteen strains of Enterobacteriaceae were sent to 62 participating centres throughout Spain; strains harboured GyrA/ParC modifications, reduced permeability and/or plasmid-mediated quinolone resistance genes. The centres were requested to evaluate nalidixic acid and five quinolones, provide raw/interpreted clinical categories and to detect/infer resistance mechanisms. Consensus results from reference centres were used to assign minor, major and very major errors (mEs, MEs and VMEs, respectively). RESULTS: Susceptibility testing in the participating centres was frequently performed using the MicroScan WalkAway, Vitek 2 and Wider systems (48%, 30% and 8%, respectively). CLSI/EUCAST breakpoints were used in 71%/29% of the determinations. The percentage of VMEs for all quinolones was well below 2%. Only ofloxacin and moxifloxacin showed higher values for raw VMEs (6.6%), which decreased to 0% and 2.9%, respectively, in the interpreted VMEs. These errors were particularly associated with the CC-03 strain [qnrS2 + aac(6')-Ib-cr]. For MEs, percentages were always <10%, except in the case of ofloxacin and nalidixic acid. There was a significantly higher percentage of all types of errors for strains whose MICs were at the border of clinical breakpoints. CONCLUSIONS: The use of different breakpoints and methods, the complexity of mutation-driven and transferable resistance mechanisms and the absence of specific tests for detecting low-level resistance lead to high variability and represent a challenge to accuracy in susceptibility testing, particularly in strains with MICs on the border of clinical breakpoints.

Interplay between plasmid-mediated and chromosomal-mediated fluoroquinolone resistance and bacterial fitness in Escherichia coli.

OBJECTIVES: The aim of this study was to analyse the interplay among plasmid-mediated qnr genes, alone or in combination with multiple chromosomal-mediated fluoroquinolone (FQ) resistance determinants, susceptibility to FQs and bacterial fitness in an isogenic Escherichia coli collection. METHODS: E. coli ATCC 25922 was used to modify or delete chromosomal genes. qnr genes were cloned into the pBK-CMV vector. The MICs of FQs were determined by microdilution. Mutant prevention concentration and frequency of mutants were evaluated. Bacterial fitness was analysed using ΔlacZ system competition assays using in vitro and in vivo models. RESULTS: The relationships between the number of resistance mutations and bacterial fitness were complex. With specific combinations of resistance mechanisms the addition of a new resistance mutation was shown to improve bacterial fitness. qnrA1 caused a decrease in fitness (7%-21%) while qnrS1 caused an increase in fitness (9%-21%) when combined with chromosomal mutations. We identified susceptible triple mutants in which the acquisition of a fourth resistance mutation significantly increased fitness and at the same time reached the clinical resistance level (the acquisition of qnrS1 in a S83L + D87N + ΔmarR genetic background). A strong correlation with the production of reactive oxygen species, as well as changes in susceptibility, was observed following treatment with ciprofloxacin. CONCLUSIONS: Our data indicate that there may be critical stages (depending on the genotype) in resistance development, including chromosomal- and plasmid-mediated mechanisms, at which some low-fitness mutants below the resistance breakpoint are able to evolve clinical resistance with just one or two mutations, and show increased fitness.

Impact of suppression of the SOS response on protein expression in clinical isolates of Escherichia coli under antimicrobial pressure of ciprofloxacin.

Introduction/objective: Suppression of the SOS response in combination with drugs damaging DNA has been proposed as a potential target to tackle antimicrobial resistance. The SOS response is the pathway used to repair bacterial DNA damage induced by antimicrobials such as quinolones. The extent of lexA-regulated protein expression and other associated systems under pressure of agents that damage bacterial DNA in clinical isolates remains unclear. The aim of this study was to assess the impact of this strategy consisting on suppression of the SOS response in combination with quinolones on the proteome profile of Escherichia coli clinical strains. Materials and methods: Five clinical isolates of E. coli carrying different chromosomally- and/or plasmid-mediated quinolone resistance mechanisms with different phenotypes were selected, with E. coli ATCC 25922 as control strain. In addition, from each clinical isolate and control, a second strain was created, in which the SOS response was suppressed by deletion of the recA gene. Bacterial inocula from all 12 strains were then exposed to 1xMIC ciprofloxacin treatment (relative to the wild-type phenotype for each isogenic pair) for 1 h. Cell pellets were collected, and proteins were digested into peptides using trypsin. Protein identification and label-free quantification were done by liquid chromatography-mass spectrometry (LC-MS) in order to identify proteins that were differentially expressed upon deletion of recA in each strain. Data analysis and statistical analysis were performed using the MaxQuant and Perseus software. Results: The proteins with the lowest expression levels were: RecA (as control), AphA, CysP, DinG, DinI, GarL, PriS, PsuG, PsuK, RpsQ, UgpB and YebG; those with the highest expression levels were: Hpf, IbpB, TufB and RpmH. Most of these expression alterations were strain-dependent and involved DNA repair processes and nucleotide, protein and carbohydrate metabolism, and transport. In isolates with suppressed SOS response, the number of underexpressed proteins was higher than overexpressed proteins. Conclusion: High genomic and proteomic variability was observed among clinical isolates and was not associated with a specific resistant phenotype. This study provides an interesting approach to identify new potential targets to combat antimicrobial resistance.

Group IIC intron with an unusual target of integration in Enterobacter cloacae.

A potential role of group IIC-attC introns in integron gene cassette formation, that is, the way in which they could provide the attC sequence essential for recombination, has been proposed. Group IIC introns usually target the attC site of gene cassettes and more specifically their inverse core. Here we characterized a novel group IIC intron targeting the core site of the aadA1 gene cassette attC site (aadA1-qacEΔ1 gene cassette junction) from enterobacterial isolates. Intron mobility (retrohoming) was analyzed using a two-plasmid assay performed in Escherichia coli. Intron mobility assays confirmed the mobilization-integration of the group II intron into the core site of the aadA2, bla(VIM-2), bla(CARB-2), aac(6')-Ib, dfrXVb, arr2, cmlA4, and aadB gene cassettes but not into the attI site. This mobility was dependent on maturase activity. Reverse transcriptase PCR showed that this intron was transcriptionally active, and an intermediate circular form was detected by inverse PCR. This element was linked to the bla(VEB-1) extended-spectrum ß-lactamase gene in a high number of enterobacterial isolates. A phylogenetic tree showed that the identified element was located in a branch separate from group IIC-attC introns, being an IIC intron possessing the ability to integrate using the core site of the attC sites as target.

Prevalence, Incidence, and Risk Factors for Intestinal Colonization Due to Fluoroquinolone-Resistant ST131 Escherichia coli: a Longitudinal Study in Highly Dependent, Long-Term Care Facility Residents.

Escherichia coli ST131 clade C is an important driver for fluoroquinolone resistance (FQ-R). We conducted a prospective observational study in residents from two long-term care facilities (LTCFs) in Seville, Spain, in 2018. Fecal swabs and environmental samples were obtained. E. coli isolates were screened for clade C, FQ-R ST131 by PCR, and molecular typing by PFGE; representatives from pulsotypes were studied by whole-genome-sequencing (WGS) and assigned to lineages (cgSTs). Prevalence of colonization at each time point, incidence density, and risk factors for acquisition were studied. Seventy-six FQ-R ST131 E. coli isolates belonging to 34 cgSTs were obtained; 24 belonging to subclade C1 (116 isolates, 65.9%) and 10 to C2 (60, 34.1%). C1 lineages showed lower virulence scores than C2 (median [IQR], 19 [18 to 20] versus 21 [20 to 21.5], P = 0.001) and higher number of plasmids (4 [3 to 5] versus 2 [2 to 3], P = 0.01). aac(6')-Ib-cr and blaOXA-1 were less frequent in C1 than C2 (2 [8.3%] versus 6 [60%], P = 0.003 for both); ESBL genes were detected in eight (33.3%) C1 (5 blaCTX-M-27) and three (30%) C2 (all blaCTX-M-15). Of the 82 residents studied, 49 were colonized at some point (59.7%), with a pooled prevalence of 38.6%. Incidence density of new lineage acquisition was 2.22 per 100 resident weeks (1.28 and 0.93 C1 and C2 subclades, respectively). Independent risk factors for acquisitions were having a colonized roommate (HR = 4.21; 95% CI = 1.71 to 10.36; P = 0.002) and urinary or fecal incontinence (HR = 2.82; 95% CI = 1.21 to 6.56; P = 0.01). LTCFs are important reservoirs of clade C ST131 E. coli. The risk factors found suggest that cross-transmission is the most relevant transmission mechanisms. IMPORTANCE We aimed at investigating the microbiological and epidemiological features of clade C fluoroquinolone-resistant ST131 E. coli isolates colonizing highly dependent residents in long-term care facilities (LTCFs) during 40 weeks and the risk factors of acquisition. Isolates from C1 and C2 subclades were characterized in this environment. The clonality of the isolates was characterized and they were assigned to lineages (cgSTs), Resistance genes, virulence factors, and plasmids were also described. This study suggests that cross-transmission is the most relevant transmission mechanisms; however, environmental colonization might also play a role. We believe the data provide useful information to depict the epidemiology of these bacteria by merging detailed microbiological and epidemiological information.

Effect of Glycerol on Fosfomycin Activity against Escherichia coli.

Fosfomycin is an antimicrobial that inhibits the biosynthesis of peptidoglycan by entering the bacteria through two channels (UhpT and GlpT). Glycerol is clinically used as a treatment for elevated intracranial pressure and induces the expression of glpT in Escherichia coli. Glycerol might offer synergistic activity by increasing fosfomycin uptake. The present study evaluates the use of glycerol at physiological concentrations in combination with fosfomycin against a collection of isogenic mutants of fosfomycin-related genes in E. coli strains. Induction of fosfomycin transporters, susceptibility tests, interaction assays, and time-kill assays were performed. Our results support the notion that glycerol allows activation of the GlpT transporter, but this induction is delayed over time and is not homogeneous across the bacterial population, leading to contradictory results regarding the enhancement of fosfomycin activity. The susceptibility assays showed an increase in fosfomycin activity with glycerol in the disk diffusion assay but not in the agar dilution or broth microdilution assays. Similarly, in the time-kill assays, the effect of glycerol was absent by the emergence of fosfomycin-resistant subpopulations. In conclusion, glycerol may not be a good candidate for use as an adjuvant with fosfomycin.