Jesús Machuca

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.

Exposure to diverse antimicrobials induces the expression of qnrB1, qnrD and smaqnr genes by SOS-dependent regulation

OBJECTIVES Direct SOS-dependent regulation of qnrB genes by fluoroquinolones mediated by LexA was reported. The smaqnr gene, on the Serratia marcescens chromosome, and qnrD both contain a putative LexA box. The aim of this study was to evaluate whether smaqnr or qnrD genes are induced via SOS-dependent mechanisms, and to investigate whether other antimicrobial agents induce qnrB, qnrD and smaqnr expression. METHODS RT-PCR was used to evaluate qnrB1, qnrD and smaqnr expression. Different concentrations of ciprofloxacin, levofloxacin, moxifloxacin and ceftazidime were evaluated as inducers. Additionally, the promoter regions of qnrB1, qnrD and smaqnr were fused transcriptionally to green fluorescent protein and used in reporter gene assays. Disc diffusion assays with different antimicrobial agents were used to detect induction. Measurements of transcriptional induction by ciprofloxacin were carried out using a plate reader. RESULTS RT-PCR assays showed that qnrB1, qnrD and smaqnr were induced at different concentrations of ciprofloxacin, moxifloxacin, levofloxacin and ceftazidime, increasing transcription 1.5- to 16.3-fold compared with basal expression, and depending on the antimicrobial agent and promoter analysed. The reporter gene assays showed that the qnrB1, qnrD and smaqnr genes were induced by ciprofloxacin, as expected, but also by ceftazidime, ampicillin and trimethoprim in Escherichia coli wild-type strains, but not in the recA-deficient E. coli HB101. Induction was not evident for imipenem or gentamicin. CONCLUSIONS β-Lactams and trimethoprim, along with fluoroquinolones, induce transcription of qnrB, qnrD and smaqnr genes using SOS-dependent regulation. These results show the direct SOS-dependent regulation of a low-level fluoroquinolone resistance mechanism in response to other antimicrobials.

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.

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.

First report of the plasmid-mediated colistin resistance gene mcr-1 in a clinical Escherichia coli isolate in Algeria.

Infections caused by Gram-negative bacteria pose an immense challenge due to limited treatment options. Older agents such as polymyxins and fosfomycin, which were rarely used in the past due to efficacy and/or toxicity concerns, along with the more recent tigecycline have become last-resort choices. None the less, acquired resistance to polymyxins is increasingly reported in Enterobacteriaceae, including Escherichia coli [1]. Emergence of the plasmid-mediated colistin resistance genemcr1, encoding a phosphoethanolamine transferase, was first described in Enterobacteriaceae isolates by Liu et al [2]; this transmissible gene was initially identified in E. coli and Klebsiella pneumoniae isolates recovered between 2011 and 2014 from animal (farm pigs, retail pork and chicken meat) and human sources in several Chinese regions. The mcr-1 gene has in fact been described in different parts of the world in Enterobacteriaceae isolates both from animals and the environment, and sporadically from humans [1,3]. In Algeria, a previous study showed the presence of this gene in an E. coli strain recovered from chicken in 2015 [4]. Here we report a clinical E. coli strain (O29) resistant to colistin according to Clinical and Laboratory Standards Institute (CLSI) criteria [minimum inhibitory concentration (MIC) of 8mg/L] recovered from the sperm culture of a 29-year-old man admitted to the University Hospital of Oran, in Oran, northwestern Algeria, for infertility in September 2011. Susceptibility testing showed that the strain was also resistant to cephalosporins, tetracycline and fluoroquinolones but remained susceptible to carbapenems, aminoglycosides, trimethoprim/ sulphamethoxazole and tigecycline. Furthermore, PCR and sequencing [2] revealed that the isolate harboured both the mcr-1 gene and the blaCTX-M-15 extended-spectrum β-lactamase (ESBL) gene. Multilocus sequence typing (MLST) identified E. coliO29 as belonging to ST405. To our knowledge, this is the first description of the mcr-1 gene in this uropathogenic sequence type. Kieser extraction and Southern blot analysis showed that the mcr-1 gene was located on a plasmid of ca. 140 kb. Analysis of plasmid incompatibility groups revealed that the plasmid belonged to the IncFIB group. The first plasmid coding for the mcr-1 gene was associated with an IncI2-type backbone [2] and to our knowledge this is the first association of the mcr-1 gene with this incompatibility group. Mating-out assays were performed and showed that the plasmid carrying the mcr-1 gene was mobilised by conjugation to the azide-resistant E. coli J53 recipient at a frequency of 10–7 cells/recipient. The transconjugants obtainedwere resistant to colistin with MICs of 4 mg/L, and there was no association with other antibiotic markers. Previous reports, however, have shown the mcr-1 gene and an ESBL gene co-resident on a single plasmid [5]. The origin of this E. coli strain as well as its relationship with a possible animal reservoir remains unknown. None the less, an animal origin of the strain would be indicated on the basis of the widespread use of colistin in veterinary medicine, helping to select for colistin resistance. This reinforces the idea that antibiotic resistance issues should be considered using the ‘OneWorld, One Health’ approach. This is the first case of MCR-1 in a human clinical isolate in Algeria. These findings highlight the transferability of colistin resistance, which constitutes a real danger to public health. Funding: This work was supported by the Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III [projects PI11-00934 and PI14/00940] and the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía [P11-CTS-7730], Spain, by the Plan Nacional de I +D + i 2008–2011 and the Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Economía y Competitividad, the Spanish Network for Research in Infectious Diseases (REIPI RD12/0015)—co-financed by European Development Regional Fund ‘A way to achieve Europe’ ERDF. Competing interests: None declared. Ethical approval: Not required.

Lactococcus garvieae carries a chromosomally encoded pentapeptide repeat protein that confers reduced susceptibility to quinolones in Escherichia coli producing a cytotoxic effect.

This study characterises a chromosomal gene of Lactococcus garvieae encoding a pentapeptide repeat protein designated as LgaQnr. This gene has been implicated in reduced susceptibility to quinolones in this bacterium, which is of relevance to both veterinary and human medicine. All of the L. garvieae isolates analysed were positive for the lgaqnr gene. The expression of lgaqnr in Escherichia coli reduced the susceptibility to quinolones, producing an adverse effect. The reduced susceptibility to ciprofloxacin was 16-fold in E. coli ATCC 25922 and 32-fold in E. coli DH10B, compared to the control strains. The minimum inhibitory concentration of nalidixic acid was also increased 4 or 5-fold. The effect of the expression of lgaqnr in E. coli was investigated by electron microscopy and was observed to affect the structure of the cell and the inner membrane of the recombinant cells.

Characterization of an outbreak due to CTX-M-15-producing Klebsiella pneumoniae lacking the blaOXA-48 gene belonging to clone ST405 in a neonatal unit in southern Spain

Unidad intercentros de Enfermedades Infecciosas, Microbiologı́a y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain; Unidad de Microbiologı́a Clı́nica, Hospital Universitario Reina Sofı́a-IMIBIC-Universidad de Cordoba, Cordoba, Spain; Unidad de Microbiologı́a Clı́nica, Hospital Universitario La Paz-IdiPaz, Madrid, Spain; Department of Microbiology, University of Seville, Seville, Spain

Staphylococcus aureus carriage in older populations in community residential care homes: Prevalence and molecular characterization of MRSA isolates

INTRODUCTION The epidemiology of S. aureus depends on conditions in specific populations. Few studies of S. aureus colonization in the older population have been performed in Spain. The aim of this study was to determine the prevalence of methicillin-resistant S. aureus (MRSA) colonization and its molecular epidemiological characteristics in an institutionalized population in community residential care homes in Cadiz, Spain. METHODS A cross-sectional epidemiological study was conducted in three residential care homes for older people. Axilla and nostril samples were tested. Identification of S. aureus and antimicrobial susceptibility testing were by MALDI-TOF and MicroScan panels. MRSA strains were subjected to SCCmec typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The presence of Panton-Valentine leukocidin (PVL) genes was determined by PCR in all S. aureus strains. RESULTS A total of 293 residents were included. Fifty-one residents (17.4%) were colonized with methicillin-sensitive S. aureus (MSSA) and 11 (3.8%) with MRSA. Resistance to at least two aminoglycosides was observed in 25.4% of MSSA and 90.9% and of MRSA isolates, and resistance to levofloxacin in 80.3% of MSSA and 100% of MRSA isolates. SCCmecIV was detected in all isolates and all except one (ST-125) were ST-8. None of the S. aureus isolates were positive for PVL. CONCLUSIONS A low rate of S. aureus carriage was detected and the prevalence of MRSA was very low. ST8-MRSA-IVc was the dominant clone, and only one strain belonged to ST125-MRSA-IVc. We found MRSA transmission within the residential care homes and a very high rate of quinolone resistance in MSSA and MRSA.