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

Role of crumb tyre aggregates in rubberised concrete contained granulated blast-furnace slag

Wise management of waste materials can be quite intensive in terms of ecological friendliness and human safety. Proper recycling of industrial wastes can lead to immense practical benefits. This paper determines the feasibility of improving the properties of concrete by incorporating waste tyre aggregates (TAs) and ground granulated blast-furnace slag (GGBFS) at varied percentages. Construction applications of rubberised concrete (RC) is limited due to presence of weak bonds between the cement paste and rubber lumps. The size and hardness of the rubber aggregates in RC make its compressive strength lower compared to traditional one. To overcome such shortcomings, GGBFS was included. Furthermore, crumb TAs of two different size distributions (fine lumps of size range 1 to 4 mm and coarse piles of sizes within 5 to 8 mm) at varied levels (5, 10, 20 and 30% of volume) were used to replace GGBFS addition to concrete. Three groups of concrete specimens were prepared, first group using TAs (size 1 to 4 mm) as partially replaced fine aggregates, second group using TAs (size 5 to 8 mm) as partly replaced coarse aggregates and the last group partially replaced both TAs. Next, GGBFS of 20% as partial replacement of cement was admixed to all groups and compared with the control mix. Workability and mechanical properties of the designed mixes were evaluated in terms of slump and compacting factor, compressive, flexural and tensile strengths, and modulus of elasticity. Specimens containing GGBFS revealed improved mechanical behaviour. Meanwhile, the compressive strength of OPC specimen (after 3 months of curing) was 37.17 MPa. Moreover, specimen mixed with 5% of TA as replacement of coarse aggregates and 20% of GGBFS was almost 6% higher than the OPC specimens (39.54 MPa). It was concluded that recycled TAs as waste material could potentially be combined with GGBFS to attain high strength RC, constituting a novel strategy with immeasurable environmental, technological and economic benefits

Incomplete hippocampalInversion : a comprehensive MRI study of over 2000 subjects

The incomplete-hippocampal-inversion (IHI), also known as malrotation, is an atypical anatomical pattern of the hippocampus, which has been reported in healthy subjects in different studies. However, extensive characterization of IHI in a large sample has not yet been performed. Furthermore, it is unclear whether IHI are restricted to the medial-temporal lobe or are associated with more extensive anatomical changes. Here, we studied the characteristics of IHI in a community-based sample of 2008 subjects of the IMAGEN database and their association with extra-hippocampal anatomical variations. The presence of IHI was assessed on T1-weighted anatomical magnetic resonance imaging (MRI) using visual criteria. We assessed the association of IHI with other anatomical changes throughout the brain using automatic morphometry of cortical sulci. We found that IHI were much more frequent in the left hippocampus (left: 17%, right: 6%, χ2−test, p < 10−28). Compared to subjects without IHI, subjects with IHI displayed morphological changes in several sulci located mainly in the limbic lobe. Our results demonstrate that IHI are a common left-sided phenomenon in normal subjects and that they are associated with morphological changes outside the medial temporal lobe

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

A Comprehensive MRI Study of Over 2000 Subjects

The incomplete-hippocampal-inversion (IHI), also known as malrotation, is an atypical anatomical pattern of the hippocampus, which has been reported in healthy subjects in different studies. However, extensive characterization of IHI in a large sample has not yet been performed. Furthermore, it is unclear whether IHI are restricted to the medial-temporal lobe or are associated with more extensive anatomical changes. Here, we studied the characteristics of IHI in a community-based sample of 2008 subjects of the IMAGEN database and their association with extra-hippocampal anatomical variations. The presence of IHI was assessed on T1-weighted anatomical magnetic resonance imaging (MRI) using visual criteria. We assessed the association of IHI with other anatomical changes throughout the brain using automatic morphometry of cortical sulci. We found that IHI were much more frequent in the left hippocampus (left: 17%, right: 6%, χ2−test, p < 10−28). Compared to subjects without IHI, subjects with IHI displayed morphological changes in several sulci located mainly in the limbic lobe. Our results demonstrate that IHI are a common left-sided phenomenon in normal subjects and that they are associated with morphological changes outside the medial temporal lobe

Analyse de la résistance aux antibiotiques chez les entérobactéries et étude d’une potentielle voie alternative aux traitements antibiotiques

The increase of multidrug-resistant bacteria (BMR) to antibiotics is a major public health problem. The first objective of the thesis was to search for the presence of BMR, poorly documented in the community in Tunisia. For the first time, we isolate a Klebsiella pneumoniae belonging to the sequence type ST147 producing carbapenemase NDM-1 in this setting, at Sfax. Our data also indicate an unusually high proportion (47%) of Escherichia coli producing committally two extended-spectrum ß-lactamases. Four of them, with CTX-M-15 and CTX-M-27, are divided into 2 clonal strains of type A-ST617 (2 isolates) and B2-ST131 subclade C2 (2 isolates). All contain a plasmid with the same allelic combination, F31: A4: B1; suggesting a possible dissemination of this replicon. In another study (community-based, Djerba), a multiresistant clonal strain Eh22 of Enterobacter hormaechei, containing a 300 kbp conjugative plasmid of IncHI2, was isolated from 2 patients without apparent epidemiological relationship. The plasmid was sequenced and shows the presence of different resistance genes including 4 genes encoding β-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 and blaSHV-12). In a second part, we studied in Eh22, the resistance to colistin (CS), antibiotic of last resort. After selection of an in vitro mutant, we showed for the first time, in Enterobacter spp, that this resistance can be due to a mutation in the gene encoding MgrB, a negative regulator of the 2-component PhoQP system that allows the synthesis of cationic residues on lipopolysaccharide, target of CS. In the last part, bacteriocins-like molecules active on BMR were searched in a collection of Bacillus thurengiensis. One of them, BUPM103, inhibits the growth of BMR. The gene for a potential bacthuricin F103 (11 kDa) was identified by an in silico analysis and it was produced in E. coli. The filtered supernatant secretion showed a growth inhibitory activity against a multiresistant K. pneumoniae, in contrast to control (without secretion). This recombinant bacthuricine could constitute a therapeutic alternative for the BMR treatment.L’augmentation des bactéries multirésistantes (BMR) aux antibiotiques est un problème majeur de santé publique. Le premier objectif de la thèse a été de rechercher la présence, peu documentée, de BMR dans la communauté en Tunisie. Pour la première fois, nous isolons une Klebsiella pneumoniae de séquence type ST147 productrice de la carbapénèmase NDM-1 dans ce milieu, à Sfax. Nos données indiquent aussi une proportion inhabituellement élevée (47%) d’Escherichia coli produisant deux ß-lactamases à spectre élargi. Quatre d’entre eux, avec CTX-M-15 et CTX-M-27, se divisent en 2 souches clonales de type A-ST617 (2 isolats) et B2-ST131 subclade C2 (2 isolats). Toutes contiennent un plasmide avec la même combinaison allélique, F31:A4:B1 ; suggérant une possible dissémination de ce réplicon. Lors d’une autre étude (milieu communautaire, Djerba), une souche clonale Eh22 d’Enterobacter hormaechei multirésistante, contenant un plasmide conjugatif IncHI2 de 300 kpb, a été isolée chez 2 patients sans lien épidémiologique apparent. Le plasmide a été séquencé et montre la présence de différents gènes de résistance incluant 4 gènes codant pour des ß-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 et blaSHV-12). Dans une seconde partie, nous avons étudié chez Eh22 la résistance à la colistine (CS), antibiotique de dernier recours. Après avoir sélectionné un mutant in vitro, nous avons montré pour la première fois, chez Enterobacter spp, que cette résistance pouvait être due à une mutation dans le gène codant pour MgrB, un régulateur négatif du système à 2 composants PhoQP qui permet la synthèse de groupements cationiques sur le lipopolysaccharide, cible de la CS. Dans une dernière partie, des molécules de type bactériocines actives sur les BMR ont été recherchées à partir d’une collection de Bacillus thurengiensis. L’une d’elle, BUPM103, inhibe la croissance de BMR. Une analyse in silico a permis d’identifier le gène d’une potentielle bacthuricine F103 (11 kDa) qui a été produite dans E. coli. Le surnageant de sécrétion filtré a montré une activité d’inhibition de la croissance de K. pneumoniae multirésistante, contrairement au contrôle (sans sécrétion). Cette bacthuricine recombinante pourrait constituer une alternative thérapeutique pour le traitement des BMR

Thermal Conductivity of Coconut Shell-Incorporated Concrete: A Systematic Assessment via Theory and Experiment

To minimize the energy consumption and adverse impact of excessive waste accumulation on the environment, coconut shell (CA) became a potential (partial) replacement agent for fine aggregates in structural concrete production. Thus, systematic experimental and theoretical studies are essential to determine the thermal and structural properties of such concrete containing optimum level of CA. In this view, an artificial neural network (ANN) model, gene expression programming (GEP) model, and response surface method (RS) were used to predict and optimize the desired engineering characteristics of some concrete mixes designed with various levels of CA inclusion. Furthermore, the proposed model&rsquo;s performance was assessed in terms of different statistical parameters calculated using ANOVA. The results revealed that the proposed concrete mix made using 53% of CA as a partial replacement of fine aggregate achieved an optimum density of 2246 kg/m3 and thermal conductivity of 0.5952 W/mK, which was lower than the control specimen (0.79 W/mK). The p-value of the optimum concrete mix was less than 0.0001 and the F-value was over 147.47, indicating the significance of all models. It is asserted that ANN, GEP, and RSM are accurate and reliable, and can further be used to predict a strong structural&ndash;thermal correlation with minimal error. In brief, the specimen composed with 53% of CA as a replacement for fine aggregate may be beneficial to develop environmentally amiable green structural concrete

Analysis of antibiotic resistance in enterobacteria and study of a potential alternative way to antibiotic treatments

L’augmentation des bactéries multirésistantes (BMR) aux antibiotiques est un problème majeur de santé publique. Le premier objectif de la thèse a été de rechercher la présence, peu documentée, de BMR dans la communauté en Tunisie. Pour la première fois, nous isolons une Klebsiella pneumoniae de séquence type ST147 productrice de la carbapénèmase NDM-1 dans ce milieu, à Sfax. Nos données indiquent aussi une proportion inhabituellement élevée (47%) d’Escherichia coli produisant deux ß-lactamases à spectre élargi. Quatre d’entre eux, avec CTX-M-15 et CTX-M-27, se divisent en 2 souches clonales de type A-ST617 (2 isolats) et B2-ST131 subclade C2 (2 isolats). Toutes contiennent un plasmide avec la même combinaison allélique, F31:A4:B1 ; suggérant une possible dissémination de ce réplicon. Lors d’une autre étude (milieu communautaire, Djerba), une souche clonale Eh22 d’Enterobacter hormaechei multirésistante, contenant un plasmide conjugatif IncHI2 de 300 kpb, a été isolée chez 2 patients sans lien épidémiologique apparent. Le plasmide a été séquencé et montre la présence de différents gènes de résistance incluant 4 gènes codant pour des ß-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 et blaSHV-12). Dans une seconde partie, nous avons étudié chez Eh22 la résistance à la colistine (CS), antibiotique de dernier recours. Après avoir sélectionné un mutant in vitro, nous avons montré pour la première fois, chez Enterobacter spp, que cette résistance pouvait être due à une mutation dans le gène codant pour MgrB, un régulateur négatif du système à 2 composants PhoQP qui permet la synthèse de groupements cationiques sur le lipopolysaccharide, cible de la CS. Dans une dernière partie, des molécules de type bactériocines actives sur les BMR ont été recherchées à partir d’une collection de Bacillus thurengiensis. L’une d’elle, BUPM103, inhibe la croissance de BMR. Une analyse in silico a permis d’identifier le gène d’une potentielle bacthuricine F103 (11 kDa) qui a été produite dans E. coli. Le surnageant de sécrétion filtré a montré une activité d’inhibition de la croissance de K. pneumoniae multirésistante, contrairement au contrôle (sans sécrétion). Cette bacthuricine recombinante pourrait constituer une alternative thérapeutique pour le traitement des BMR.The increase of multidrug-resistant bacteria (BMR) to antibiotics is a major public health problem. The first objective of the thesis was to search for the presence of BMR, poorly documented in the community in Tunisia. For the first time, we isolate a Klebsiella pneumoniae belonging to the sequence type ST147 producing carbapenemase NDM-1 in this setting, at Sfax. Our data also indicate an unusually high proportion (47%) of Escherichia coli producing committally two extended-spectrum ß-lactamases. Four of them, with CTX-M-15 and CTX-M-27, are divided into 2 clonal strains of type A-ST617 (2 isolates) and B2-ST131 subclade C2 (2 isolates). All contain a plasmid with the same allelic combination, F31: A4: B1; suggesting a possible dissemination of this replicon. In another study (community-based, Djerba), a multiresistant clonal strain Eh22 of Enterobacter hormaechei, containing a 300 kbp conjugative plasmid of IncHI2, was isolated from 2 patients without apparent epidemiological relationship. The plasmid was sequenced and shows the presence of different resistance genes including 4 genes encoding β-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 and blaSHV-12). In a second part, we studied in Eh22, the resistance to colistin (CS), antibiotic of last resort. After selection of an in vitro mutant, we showed for the first time, in Enterobacter spp, that this resistance can be due to a mutation in the gene encoding MgrB, a negative regulator of the 2-component PhoQP system that allows the synthesis of cationic residues on lipopolysaccharide, target of CS. In the last part, bacteriocins-like molecules active on BMR were searched in a collection of Bacillus thurengiensis. One of them, BUPM103, inhibits the growth of BMR. The gene for a potential bacthuricin F103 (11 kDa) was identified by an in silico analysis and it was produced in E. coli. The filtered supernatant secretion showed a growth inhibitory activity against a multiresistant K. pneumoniae, in contrast to control (without secretion). This recombinant bacthuricine could constitute a therapeutic alternative for the BMR treatment

Analysis of antibiotic resistance in enterobacteria and study of a potential alternative way to antibiotic treatments

L’augmentation des bactéries multirésistantes (BMR) aux antibiotiques est un problème majeur de santé publique. Le premier objectif de la thèse a été de rechercher la présence, peu documentée, de BMR dans la communauté en Tunisie. Pour la première fois, nous isolons une Klebsiella pneumoniae de séquence type ST147 productrice de la carbapénèmase NDM-1 dans ce milieu, à Sfax. Nos données indiquent aussi une proportion inhabituellement élevée (47%) d’Escherichia coli produisant deux ß-lactamases à spectre élargi. Quatre d’entre eux, avec CTX-M-15 et CTX-M-27, se divisent en 2 souches clonales de type A-ST617 (2 isolats) et B2-ST131 subclade C2 (2 isolats). Toutes contiennent un plasmide avec la même combinaison allélique, F31:A4:B1 ; suggérant une possible dissémination de ce réplicon. Lors d’une autre étude (milieu communautaire, Djerba), une souche clonale Eh22 d’Enterobacter hormaechei multirésistante, contenant un plasmide conjugatif IncHI2 de 300 kpb, a été isolée chez 2 patients sans lien épidémiologique apparent. Le plasmide a été séquencé et montre la présence de différents gènes de résistance incluant 4 gènes codant pour des ß-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 et blaSHV-12). Dans une seconde partie, nous avons étudié chez Eh22 la résistance à la colistine (CS), antibiotique de dernier recours. Après avoir sélectionné un mutant in vitro, nous avons montré pour la première fois, chez Enterobacter spp, que cette résistance pouvait être due à une mutation dans le gène codant pour MgrB, un régulateur négatif du système à 2 composants PhoQP qui permet la synthèse de groupements cationiques sur le lipopolysaccharide, cible de la CS. Dans une dernière partie, des molécules de type bactériocines actives sur les BMR ont été recherchées à partir d’une collection de Bacillus thurengiensis. L’une d’elle, BUPM103, inhibe la croissance de BMR. Une analyse in silico a permis d’identifier le gène d’une potentielle bacthuricine F103 (11 kDa) qui a été produite dans E. coli. Le surnageant de sécrétion filtré a montré une activité d’inhibition de la croissance de K. pneumoniae multirésistante, contrairement au contrôle (sans sécrétion). Cette bacthuricine recombinante pourrait constituer une alternative thérapeutique pour le traitement des BMR.The increase of multidrug-resistant bacteria (BMR) to antibiotics is a major public health problem. The first objective of the thesis was to search for the presence of BMR, poorly documented in the community in Tunisia. For the first time, we isolate a Klebsiella pneumoniae belonging to the sequence type ST147 producing carbapenemase NDM-1 in this setting, at Sfax. Our data also indicate an unusually high proportion (47%) of Escherichia coli producing committally two extended-spectrum ß-lactamases. Four of them, with CTX-M-15 and CTX-M-27, are divided into 2 clonal strains of type A-ST617 (2 isolates) and B2-ST131 subclade C2 (2 isolates). All contain a plasmid with the same allelic combination, F31: A4: B1; suggesting a possible dissemination of this replicon. In another study (community-based, Djerba), a multiresistant clonal strain Eh22 of Enterobacter hormaechei, containing a 300 kbp conjugative plasmid of IncHI2, was isolated from 2 patients without apparent epidemiological relationship. The plasmid was sequenced and shows the presence of different resistance genes including 4 genes encoding β-lactamases (blaTEM-1, blaDHA-1, blaCTX-M-3 and blaSHV-12). In a second part, we studied in Eh22, the resistance to colistin (CS), antibiotic of last resort. After selection of an in vitro mutant, we showed for the first time, in Enterobacter spp, that this resistance can be due to a mutation in the gene encoding MgrB, a negative regulator of the 2-component PhoQP system that allows the synthesis of cationic residues on lipopolysaccharide, target of CS. In the last part, bacteriocins-like molecules active on BMR were searched in a collection of Bacillus thurengiensis. One of them, BUPM103, inhibits the growth of BMR. The gene for a potential bacthuricin F103 (11 kDa) was identified by an in silico analysis and it was produced in E. coli. The filtered supernatant secretion showed a growth inhibitory activity against a multiresistant K. pneumoniae, in contrast to control (without secretion). This recombinant bacthuricine could constitute a therapeutic alternative for the BMR treatment