MgrB Inactivation Is Responsible for Acquired Resistance to Colistin in Enterobacter hormaechei subsp. steigerwaltii

Multidrug-resistant strains belonging to the Enterobacter cloacae complex (ECC) group, and especially those belonging to clusters C-III, C-IV, and C-VIII, have increasingly emerged as a leading cause of health care-associated infections, with colistin used as one of the last lines of treatment. However, colistin-resistant ECC strains have emerged. The aim of this study was to prove that MgrB, the negative regulator of the PhoP/PhoQ two-component regulatory system, is involved in colistin resistance in ECC of cluster C-VIII, formerly referred to as Enterobacter hor-maechei subsp. steigerwaltii. An in vitro mutant (Eh22-Mut) was selected from a clinical isolate of Eh22. The sequencing analysis of its mgrB gene showed the presence of one nucleotide deletion leading to the formation of a truncated protein of six instead of 47 amino acids. The wild-type mgrB gene from Eh22 and that of a clinical strain of Klebsiella pneumoniae used as controls were cloned, and the corresponding recombinant plasmids were used for complementation assays. The results showed a fully restored susceptibility to colistin and confirmed for the first time that mgrB gene expression plays a key role in acquired resistance to colistin in ECC strains

First description of NDM-1-positive Klebsiella pneumoniae in the Tunisian community

First description of NDM-1-positive Klebsiella pneumoniae in the Tunisian community Sir, Multidrug-resistant bacteria, especially carbapenemase-producing Enterobacteriaceae, are a major public health-threat worldwide. As part of a collaborative monitoring programme, our laboratory at the University of Bordeaux has received a collection of multidrug-resistant bacterial strains to further characterise their β-lactamase content. They were sent from private Tunisian diagnostic laboratories and were collected from community patients suffering from urinary tract infection. In this context, multidrug-resistant isolate 18TA was collected in January 2018 in Sfax region from the urine of a 45-year old female with no previous hospitalisation during the preceding month and no history of recent foreign travel. Strain 18TA had been initially classified as Enterobacter spp. by biochemical tests (API 10S gallery). Following matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonics) and confirmation by PCR amplification and sequencing of 16S rDNA, strain 18TA was re-identified as Klebsiella pneumoniae. Multilocus sequence typing (MLST) (http:// bigsdb.pasteur.fr/klebsiella/klebsiella.html) indicated that strain 18TA belonged to Sequence Type (ST), ST147. Minimum inhibitory concentrations (MICs) of various anti-microbials were determined using a BD Phoenix TM 100 automated system (BD Diagnostic Systems, Le Pont-de-Claix, France) and the results were interpreted using BD EpiCenter TM software (BD Diagnostic Systems). The MICs for ciprofloxacin and colistin were also determined by the broth microdilution method according to European Committee on Antimicrobial Susceptibility Testing 2019 guidelines (https://www.sfm-microbiologie.org/2019/05/06/ casfm-eucast-2019-v2/). Strain 18TA was resistant to all tested β-lactams, including carbapenems (Table 1). The strain was also resistant to gentamicin, tobramycin, quinolones (nalidixic acid), fluoroquinolones (cipro-floxacin) and trimethoprim/sulfamethoxazole (SXT) and showed decreased susceptibility to tigecycline (MIC = 2 mg/mL). It remained susceptible to amikacin, fosfomycin and colistin (Table 1). The imipenem/ethylene diamine tetra-acetic acid (EDTA) combined disk diffusion test was positive since the inhibition zone increased by 7 mm with the imipenem/EDTA disk compared with the imipenem disk alone, suggesting the presence of a metallo-β-lactamase (MBL) [1]. In addition, the double-disk synergy test (between amoxicillin/clavulanic acid and broad-spectrum cepha-losporins) showed the presence of an extended-spectrum β-lactamase (ESBL)-producing phenotype (data not shown). The transferability of the β-lactam resistance determinant was assessed by conjugation assay using an azide-resistant (Az R) mutant of Escherichia coli C600 as the recipient strain. Selection was performed on Mueller-Hinton agar plates supplemented with sodium azide (300 mg/mL) and ertapenem (4 mg/mL). A transfer frequency of ca. 10-4 transconjugants per donor was observed. Comparison of MICs between the transconjugant (Tc-18TA) and its recipient strain (C600 Az R) showed increased resistance not only to the tested β-lactams but also to gentamicin, tobramycin and SXT (Table 1). Total genomic DNA of strain 18TA was screened using different multiplex PCR amplifications for various β-lactamase genes (bla TEM-like , bla SHV-like , bla OXA-1-like , bla CTX-M groups 1, 2, 9, 18 and 25, bla OXA-48-like , bla KPC and bla GES and the MBL genes bla VIM , bla IMP and bla NDM) as described previously [2]. Amplification results following agarose gel electrophoresis analysis showed the presence of group 1 bla CTX-M and bla NDM genes together with bla TEM-like , bla SHV-like and bla OXA-1-like genes. Except for the bla SHV gene that was found in strain 18TA but not in the transconjugant Tc-18TA and that was attributed to the chromosomally-encoded species-specific enzyme of K. pneumoniae, the four other β-lactamases were also found in transconjugant Tc-18TA (Table 1). Amplification of the entire bla genes was performed and subsequent sequencing ((Custom DNA sequencing; Eurofins Genomics GmbH, Ebersberg, Germany) showed the presence of the narrow-spectrum β-lactamase genes bla TEM-1B and bla OXA-1 associated with the bla CTX-M-15 ESBL gene and the bla NDM-1 MBL gene both in 18TA and Tc-18TA (Table 1). Furthermore, amplifications searching for aac(3)-IIa (gentamicin and tobramycin resistance) and sul1 and dfrA1 (sulfamethoxazole and trimetho-prim resistance, respectively) were positive both in 18TA and Tc-18TA. These genes were also present in Kp3771, a ST147 NDM-1-producing K. pneumoniae strain recently recovered from a patient hospitalised in an intensive care unit of University Hospital Tahar Sfar in Tunisia [3]. NDM-1-positive K. pneumoniae strains have been previously described in Tunisia, but only from hospitalised patients [1-5]. The current study reports the first description of K. pneumoniae carrying the carbapenemase NDM-1 in the Tunisian community http://dx

Detection of Clones B2-ST131-C2 and A-ST617 in Escherichia coli Producing Both CTX-M-15 and CTX-M-27 from Tunisian Community Patients

During a two-month period (2017–2018), 336 urine samples positive for Escherichia coli were collected from Tunisian patients. Of the 336 samples, 266 were collected from community patients and 70 from hospital settings. In all, 15 ESBL producers were identified (8 and 7, respectively) and assigned to 13 pulsotypes, including four ESBL-producing E. coli (ESBL-E) with E1 and E2 profiles (2 isolates each) from community patients. The two strains E1 were identified as B2-ST131 subclade C2 and the two isolates E2, A-ST617. The four strains carrying both CTX-M-15 and CTX-M-27, exhibited the multireplicon IncFII/F1A/F1B with the same formula F31:A4:B1. Two isolates with patterns E3 and E4 (Dice coefficient, 78.7%) isolated from community and hospital settings of two geographic areas were assigned to the emerging ST131 C1-M27 subclade and contained the replicon F1:A-:B20. The remaining ESBL-E divided into different sequence types/associated CTX-M: 2 ST131-C2/CTX-M-15 and ST744/CTX-M-55, ST617/CTM-15, ST2973/CTX-M-55, ST6448/CTX-M-15, ST224/CTX-M-15, ST1431/CTX-M-15, and ST38/CTX-M-27, one isolate each. Our study reports for the first time the presence in the Tunisian community of two clones of E. coli, including the virulent clone ST131-C2 harboring both CTX-M-15 and CTX-M-27, and confirms the spread of the emergent clone ST131-C1-M-27, notably in community urinary tract infections

Etude de l’occurrence de molécules pharmaceutiques et de leurs métabolites dans des zones côtières méditerranéennes.

International audienc

Predicted and measured concentrations of venlafaxine and its metabolites in a coastal zone receiving treated wastewaters.

International audienceParent compounds and metabolites excreted in wastewater are inconstantly eliminated in WTPs and therefore are rejected mainly in surface water. However, the coastal environment suffers from pollution generated by inland activities which discharge their wastes into the sea via streams, rivers and wastewater marine outfalls. The low levels reported in the marine environment encourage the development of adapted methodologies to estimate predicted environmental concentrations (PECs). In the present work, the occurrence of VLF and its major human metabolites was investigated at a Mediterranean coastal site directly impacted by a submarine outfall. Concentrations of VLF and its metabolites were measured in the different compartments i.e. in water with passive sampler, sediment and mussels. Predicted concentrations of VLF and its main human metabolites were estimated in seawater taking into account the sold amounts of VLF, its human metabolism and the diffusion and dilution of the compounds in the coastal zone using an adapted hydrodynamic model (MARS 3D) developed by Ifremer. In seawater, estimated concentrations were in a good agreement with measured levels. Moreover, concentrations in mussels were estimated using concentrations estimated in seawater and a bioconcentration factor (BCF) linear model described in the literature for marine mussels. Estimated concentrations in biota were then compared to the concentrations in mussels detected at the studied site. Concentrations estimated with the BCF model were overestimated compared to measured concentrations, which could be explained by a possible metabolism of these compounds by mussels, not taken into account in the estimations. Moreover, attenuation mechanisms such as sorption on suspended matter could explain a lower availability of compounds for mussels in the water column and a lower bioconcentration in organisms than those expected. Studies on sorption mechanismsand metabolism of these compounds in mussels should be further performed, in order to improve these estimations. As organisms are long-term exposed to low concentrations of pharmaceuticals, possible effects could be observed. The use of sensitive approaches, such as “omics” approaches, already used for other pollutants such as heavy metals, could help obtain this information

Predicted environmental concentrations of carbamazepine, oxcarbazepine and their main metabolites in a coastal system

Pharmaceuticals are widely released in aquatic environment through treated wastewaters. They reach coastal zone indirectly via streams or directly though marine outfalls however data concerning this contamination in coastal waters are scarce. Environmental Risk Assessment (ERA) of pharmaceuticals have been conducted mostly in surface waters and has not been performed in coastal zone. The first step of ERA is to evaluate the exposure through predictive environmental concentration (PEC) values. The aim of this study was to predict the occurrence of some pharmaceuticals in a coastal aera subjected to treated wastewater (TWW) reject through a marine outfall (Fig 2.). Among pharmaceuticals, Carbamazepine, Oxcarbazepine and their main metabolites (Fig 1.) were chosen. CBZ has been proposed as an indicator of wastewater contamination and has been already detected in Mediterranean (Munaron et al., 2011). Prediction was performed based on local pharmaceuticals consumption recording and a review of pharmacokinetics data. PECs values were estimated in TWW and at the marine outfall and compared with MECs obtained by direct quantification and with POCIS implementation

A prospective study on radiation pneumonitis following conformal radiation therapy in non-small-cell lung cancer : clinical and dosimetric factors analysis

International audienceBackground and purpose: Clinical and dosimetric prognostic factors for radiation pneumonitis (RP) have been reported after three-dimensional conformal radiotherapy (3D-CRT) in patients with non-small cell lung cancer (NSCLC). Patients and methods: Ninety-six patients who received 3D-CRT for stage IA to IIIB NSCLC were evaluated prospectively. Surgery was performed before radiation in 51 % of the patients (n = 49). RP was diagnosed six-eight weeks after 3D-CRT using the Lent-Solna classification. Factors evaluated included treatment factors such as total mean lung dose (MLD), and dose-volume histogram (DVH) thresholds for several radiation dose steps. These thresholds were originally determined from the median of the irradiated lung volume at each step. Results: Six patients could not be evaluated for RP six weeks after 3D-CRT. Of the 90 remaining patients, 40 (44%) had RP (i.e. grade greater than or equal to 1) at 6 weeks, including 7 patients (7.8%) with severe RP (grade greater than or equal to 2). Regarding the whole toxicity (grade greater than or equal to 1), age ( greater than or equal to 60 years), MLD, V20 and V30 were significantly related to RP. DVH thresholds determined for radiation doses from 20 to 40 Gy were also predictive of RP. Considering only severe RP (grade greater than or equal to 2), only MLD, V20 and V30 remained associated with increased acute pulmonary toxicity. Conclusions: In this study, dosimetric factors (MLD, V20, V30) and age ( greater than or equal to60 years) were predictive of RP regarding the whole pulmonary toxicity (grade greater than or equal to 1). In addition, thresholds from 20 to 40 Gy, based on a stratification according to the median of the percentage of irradiated lung volume, were also predictive factors. They may, therefore, help discriminate patients at high and low risk for RP. However, only MLD, V20 and V30 remained associated with severe RP (grade : 2), probably due to the small number of severe events in our series