Residual risk of Pseudomonas aeruginosa waterborne contamination in an intensive care unit despite the presence of filters at all water points-of-use.

OBJECTIVE: To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa from a water network colonized by a single genotype [sequence type (ST) 299] despite the presence of antimicrobial filters in a medical intensive care unit (ICU). METHODS: During the first 19-month period since the ICU opened, contamination of the water network was assessed monthly by collecting water upstream of the filters. Downstream water was also sampled to assess the efficiency of the filters. P. aeruginosa isolates from patients were collected and compared with the waterborne ST299 P. aeruginosa by multiplex-rep polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events by other genotypes of P. aeruginosa were also assessed. RESULTS: Overall, 1.3% of 449 samples of filtered water were positive for P. aeruginosa in inoculum, varying between 1 and 104 colony-forming units/100 mL according to the tap. All P. aeruginosa hydric isolates belonged to ST299 and displayed fewer than two single nucleotide polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in five patients showed identical profiles to the hydric ST299 clone on both multiplex-rep PCR and PFGE, and differed by an average of fewer than five SNPs, confirming the water network reservoir as the source of contamination by P. aeruginosa for 4.09% of patients. Cross-transmission events by other genotypes of P. aeruginosa were responsible for the contamination of 1.75% of patients. DISCUSSION/CONCLUSION: Antimicrobial filters are not sufficient to protect patients from waterborne pathogens when the water network is highly contaminated. A microbiological survey of filtered water may be needed in units hosting patients at risk of P. aeruginosa infections, even when all water points-of-use are fitted with filters.

QMAC-dRAST for the direct testing of antibiotic susceptibility for Enterobacterales in positive blood-culture broth: a comparison of the performances with the MicroScan system and direct disc diffusion testing methods.

OBJECTIVES: To evaluate the performances of the QMAC-dRAST GN (Gram-negative) kit for rapid antimicrobial sensitivity testing (AST) and two other methods, directly on positive blood-culture broth (PBCB), by comparison with a reference method: the MicroScan method based on broth microdilution on colonies isolated on PBCB subculture. METHODS: In total, 156 samples were collected prospectively from blood cultures positive for a Gram-negative rod. Each sample was tested with four AST techniques: (i) the QMAC dRAST GN kit, (ii) the disc diffusion (DD) method, (iii) the MicroScan method applied directly to PBCB; and (iv) MicroScan with isolates from PBCB subculture, as a reference. RESULTS: For 124 PBCB containing Enterobacterales, overall essential agreement (EA) and categorical agreement (CA) between the QMAC-dRAST on PBCB and the reference reached 95.7% and 93.5%, respectively. There were 3.0% very major errors (VME), 4.0% major errors (ME) and 2.8% minor errors (mE). A comparison of MicroScan on PBCB and the reference yielded 98.8% EA, 98.5% CA, and rates of 0.6% VME, 0.9% ME and 0.7% mE. The DD method on PBCB gave a CA of 95.8% and rates of 1.7% for VME, 2.0% for ME and 1.9% for mE. Results were obtained more rapidly for QMAC-dRAST (median of 6 h 37 min versus 18 h for the MicroScan and DD methods on PBCB). CONCLUSIONS: The QMAC-dRAST system provided rapid results well correlated with the reference method on PBCB containing Enterobacterales. Given the shorter time-to-results, the QMAC-dRAST system constitutes a fast and reliable alternative to conventional AST methods.

Evolution of fluoroquinolone-resistant Escherichia coli in the gut after ciprofloxacin treatment.

BACKGROUND: Three healthy volunteers carried similar quinolone-resistant E. coli (QREC) (pulsed field gel electrophoresis profiles) in their gut before and after 14 days ciprofloxacin treatment. Given the intensity of the selective pressure and the mutagenic properties of quinolones, we determined whether these strains had evolved at the phenotypic and/or genomic levels. MATERIAL AND METHODS: Commensal QREC from before day-0 (D0), and a month after 14 days of ciprofloxacin (D42) were compared in 3 volunteers. Growth experiments were performed; acetate levels, mutation frequencies, quinolone MICs and antibiotic tolerance were measured at D0 and D42. Genomes were sequenced and single nucleotide polymorphisms (SNPs) between D0 and D42 were analyzed using DiscoSNP and breseq methods. Cytoplasmic proteins were extracted, HPLC performed and proteins identified using X!tandem software; abundances were measured by mass spectrometry using the Spectral Counting (SC) and eXtraction Ion Chromatograms (XIC) integration methods. RESULTS: No difference was found in MICs, growth characteristics, acetate concentrations, mutation frequencies, tolerance profiles, phylogroups, O-and H-types, fimH alleles and sequence types between D0 and D42. No SNP variation was evidenced between D0 and D42 isolates for 2/3 subjects; 2 SNP variations were evidenced in one. At the protein level, very few significant protein abundance differences were identified between D0 and D42. CONCLUSION: No fitness, tolerance, metabolic or genomic evolution of commensal QREC was observed overtime, despite massive exposure to ciprofloxacin in the gut. The three strains behaved as if they had been unaffected by ciprofloxacin, suggesting that gut may act as a sanctuary where bacteria would be protected from the effect of antibiotics and survive without any detrimental effect of stress.

Microdiversity of Enterococcus faecalis isolates in cases of infective endocarditis: selection of non-synonymous mutations and large deletions is associated with phenotypic modifications.

Context: Today, infective endocarditis (IE) caused by Enterococcus faecalis represents 10% of all IE and is marked by its difficult management and the frequency of relapses. Although the precise reasons for that remain to be elucidated, the evolution of the culprit strain under selective pressure through microdiversification could be, at least in part, involved. Material and methods: To further study the in situ genetic microdiversity and its possible phenotypic manifestations in E. faecalis IE, we sequenced and compared multiple isolates from the valves, blood culture and joint fluid of five patients who underwent valvular surgery. Growth rate and early biofilm production of selected isolates were also compared. Results: By sequencing a total of 58 E. faecalis genomes, we detected a considerable genomic microdiversity, not only among strains from different anatomical origins, but also between isolates from the same studied cardiac valves. Interestingly, deletions of thousands of bases including the well-known virulence factors ebpA/B/C, and srtC, as well as other large prophage sequences containing genes coding for proteins implicated in platelet binding (PlbA and PlbB) were evidenced. The study of mutations helped unveil common patterns in genes related to the cell cycle as well as central metabolism, suggesting an evolutionary convergence in these isolates. As expected, such modifications were associated with a significant impact on the in-vitro phenotypic heterogeneity, growth, and early biofilm production. Conclusion: Genome modifications associated with phenotypic variations may allow bacterial adaptation to both antibiotic and immune selective pressures, and thus promote relapses.

Management of ocular involvement in the acute phase of Stevens-Johnson syndrome and toxic epidermal necrolysis: french national audit of practices, literature review, and consensus agreement.

Stevens-Johnson Syndrome (SJS) and toxic epidermal necrolysis (TEN) can lead to severe ophthalmologic sequelae. The main risk factor is the severity of the initial ocular involvement. There are no recommendations for ocular management during acute phase.We conducted a national audit of current practice in the 11 sites of the French reference center for toxic bullous dermatoses and a review of the literature to establish therapeutic consensus guidelines. We sent a questionnaire on ocular management practices in SJS/ TEN during acute phase to ophthalmologists and dermatologists. The survey focused on ophthalmologist opinion, pseudomembrane removal, topical ocular treatment (i.e. corticosteroids, antibiotics, antiseptics, artificial tear eye drops, vitamin A ointment application), amniotic membrane transplantation, symblepharon ring use, and systemic corticosteroid therapy for ophthalmologic indication. Nine of 11 centers responded. All requested prompt ophthalmologist consultation. The majority performed pseudomembrane removal, used artificial tears, and vitamin A ointment (8/9, 90%). Combined antibiotic-corticosteroid or corticosteroid eye drops were used in 6 centers (67%), antibiotics alone and antiseptics in 3 centers (33%). Symblepharon ring was used in 5 centers (55%) if necessary. Amniotic membrane transplantation was never performed systematically and only according to the clinical course. Systemic corticosteroid therapy was occasionally used (3/9, 33%) and discussed on a case-by-case basis.The literature about ocular management practice in SJS/ TEN during acute phase is relatively poor. The role of specific treatments such as local or systemic corticosteroid therapy is not consensual. The use of preservatives, often present in eye drops and deleterious to the ocular surface, is to be restricted. Early amniotic membrane transplantation seems to be promising.

Mortality in Escherichia coli bloodstream infections: antibiotic resistance still does not make it.

BACKGROUND: Escherichia coli bloodstream infections (BSIs) account for high mortality rates (5%-30%). Determinants of death are unclear, especially since the emergence of ESBL producers. OBJECTIVES: To determine the relative weight of host characteristics, bacterial virulence and antibiotic resistance in the outcome of patients suffering from E. coli BSI. METHODS: All consecutive patients suffering from E. coli BSI in seven teaching hospitals around Paris were prospectively included for 10 months. E. coli isolates were sequenced using Illumina NextSeq technology to determine the phylogroup, ST/ST complex (STc), virulence and antimicrobial resistance gene content. Risk factors associated with death at discharge or Day 28 were determined. RESULTS: Overall, 545 patients (mean ± SD age 68.5 ±âŸ16.5 years; 52.5% male) were included. Mean Charlson comorbidity index (CCI) was 5.6 (± 3.1); 19.6% and 12.8% presented with sepsis and septic shock, respectively. Portals of entry were mainly urinary (51.9%), digestive (41.9%) and pulmonary (3.5%); 98/545 isolates (18%) were third-generation cephalosporin resistant (3GC-R), including 86 ESBL producers. In-hospital death (or at Day 28) was 52/545 (9.5%). Factors independently associated with death were a pulmonary portal of entry [adjusted OR (aOR) 6.54, 95% CI 2.23-19.2, P = 0.0006], the iha_17 virulence gene (aOR 4.41, 95% CI 1.23-15.74, P = 0.022), the STc88 (aOR 3.62, 95% CI 1.30-10.09, P = 0.014), healthcare-associated infections (aOR 1.98, 95% CI 1.04-3.76, P = 0.036) and high CCI (aOR 1.14, 95% CI 1.04-1.26, P = 0.006), but not ESBL/3GC-R. CONCLUSIONS: Host factors, portal of entry and bacterial characteristics remain major determinants associated with mortality in E. coli BSIs. Despite a high prevalence of ESBL producers, antibiotic resistance did not impact mortality. (ClinicalTrials.gov identifier: NCT02890901.).

Local outbreak of extended-spectrum ß-lactamase SHV2a-producing Pseudomonas aeruginosa reveals the emergence of a new specific sub-lineage of the international ST235 high-risk clone.

BACKGROUND: Pseudomonas aeruginosa is a major bacterial pathogen responsible for hospital-acquired infections. Although its epidemiology is considered as non-clonal, certain international high-risk multidrug-resistant clones have been recognized. AIM: From the first report of an intra-hospital outbreak due to an SHV2a-producing P. aeruginosa strain, to describe the emergence of a new ST235-specific lineage harbouring this rare extended-spectrum ß-lactamase (ESBL). METHODS: Between May and October 2018, four patients hospitalized in the cardiovascular intensive care unit of a French teaching hospital were infected by a multidrug-resistant P. aeruginosa isolate. Serotype and antimicrobial susceptibility were tested; multi-locus sequence type (MLST), core genome MLST, and resistome were determined through whole genome sequencing. A phylogenetic analysis based on single nucleotide polymorphism was performed using available ST235 genomes. FINDINGS: The four strains were susceptible to colistin, ciprofloxacin, ceftazidime-avibactam, and ceftolozane-tazobactam. blaSHV2a was identified in each genome of this ST235-O11 serotype cluster that showed an identical cgMLST profile (0-2 out of 4162 different alleles). The phylogenic analysis of 162 ST235 genomes showed that only four other strains harboured a blaSHV2a, originating from France and USA, clustering together although being different from the outbreak strains. CONCLUSIONS: Among the ST235 P. aeruginosa strains, a sub-lineage sharing a common genetic background and harbouring the blaSHV2a ESBL seems to emerge from different locations, yielding secondary local outbreaks.