Molecular mechanisms and clonal lineages of colistin-resistant bacteria across the African continent: a scoping review.

Colistin (also known as polymyxin E), a polymyxin antibiotic discovered in the late 1940s, has recently reemerged as a last-line treatment option for multidrug-resistant infections. However, in recent years, colistin-resistant pathogenic bacteria have been increasingly reported worldwide. Accordingly, the presented review was undertaken to identify, integrate and synthesize current information regarding the detection and transmission of colistin-resistant bacteria across the African continent, in addition to elucidating their molecular mechanisms of resistance. PubMed, Google Scholar and Science Direct were employed for study identification, screening and extraction. Overall, based on the developed literature review protocol and associated inclusion/exclusion criteria, 80 studies published between 2000 and 2021 were included comprising varying bacterial species and hosts. Numerous mechanisms of colistin resistance were reported, including chromosomal mutation(s) and transferable plasmid-mediated colistin resistance (encoded by mcr genes). Perhaps unexpectedly, mcr-variants have exhibited rapid emergence and spread across most African regions. The genetic variant mcr-1 is predominant in humans, animals and the natural environment, and is primarily carried by IncHI2- type plasmid. The highest number of studies reporting the dissemination of colistin-resistant Gram-negative bacteria were conducted in the North African region.

Elucidating key plant growth-promoting (PGPR) traits in Burkholderia sp. Nafp2/4-1b (=SARCC-3049) using gnotobiotic assays and whole-genome-sequence analysis.

Burkholderia sp. Nafp2/4-1b (=SARCC-3049) is a plant growth-promoting rhizobacteria (PGPR) initially isolated from the rhizosphere of pristine grassland in South Africa, and its ability to enhance growth was previously evaluated on maize (Zea mays L.). Here, the bacterium was tested with the aim of investigating its role in improving the nodulation and growth of the forage legume lucerne (Medicago sativa L.) when it is co-inoculated with the rhizobial symbionts of this legume in the glasshouse. When the co-inoculation resulted in a statistically significant (P = 0·05) increase in the number of nodules and improved plant biomass compared with single inoculation, we sequenced and analysed its genome to gain a better understanding of the genetic determinants responsible for the observed PGPR traits. The Illumina HiSeq 2500-sequenced genome resulted in 92 scaffolds, with an N50 of 322 407 bp, a total draft genome size of 7 788 045 bp and GC content of 66·2%. Analysis of the genome sequence confirmed the presence of a number of essential genes that code for various PGPR traits. The main plant beneficial genes associated with PGPR traits in Burkholderia sp. Nafp2/4-1b include pyoverdine siderophores biosynthesis gene (PvdF); acdS that codes for 1-aminocyclopropane-1-carboxylate (ACC) deaminase; the tryptophan synthase genes involved in auxin biosynthesis (TSA1, TSB1) and the pqqABCDE operon related to phosphate solubilization. This study generated valuable information on the potential of the PGPR Burkholderia sp. strain Nafp2/4-1b as an effective commercial inoculant, which warrants further formulation and field application studies before developing it into a low cost, environmentally safe and effective biofertilizer.

Man against machine: diagnostic performance of a deep learning convolutional neural network for dermoscopic melanoma recognition in comparison to 58 dermatologists.

Background: Deep learning convolutional neural networks (CNN) may facilitate melanoma detection, but data comparing a CNN's diagnostic performance to larger groups of dermatologists are lacking. Methods: Google's Inception v4 CNN architecture was trained and validated using dermoscopic images and corresponding diagnoses. In a comparative cross-sectional reader study a 100-image test-set was used (level-I: dermoscopy only; level-II: dermoscopy plus clinical information and images). Main outcome measures were sensitivity, specificity and area under the curve (AUC) of receiver operating characteristics (ROC) for diagnostic classification (dichotomous) of lesions by the CNN versus an international group of 58 dermatologists during level-I or -II of the reader study. Secondary end points included the dermatologists' diagnostic performance in their management decisions and differences in the diagnostic performance of dermatologists during level-I and -II of the reader study. Additionally, the CNN's performance was compared with the top-five algorithms of the 2016 International Symposium on Biomedical Imaging (ISBI) challenge. Results: In level-I dermatologists achieved a mean (±standard deviation) sensitivity and specificity for lesion classification of 86.6% (±9.3%) and 71.3% (±11.2%), respectively. More clinical information (level-II) improved the sensitivity to 88.9% (±9.6%, P = 0.19) and specificity to 75.7% (±11.7%, P < 0.05). The CNN ROC curve revealed a higher specificity of 82.5% when compared with dermatologists in level-I (71.3%, P < 0.01) and level-II (75.7%, P < 0.01) at their sensitivities of 86.6% and 88.9%, respectively. The CNN ROC AUC was greater than the mean ROC area of dermatologists (0.86 versus 0.79, P < 0.01). The CNN scored results close to the top three algorithms of the ISBI 2016 challenge. Conclusions: For the first time we compared a CNN's diagnostic performance with a large international group of 58 dermatologists, including 30 experts. Most dermatologists were outperformed by the CNN. Irrespective of any physicians' experience, they may benefit from assistance by a CNN's image classification. Clinical trial number: This study was registered at the German Clinical Trial Register (DRKS-Study-ID: DRKS00013570; https://www.drks.de/drks_web/).

Feasibility of early physical therapy for dizziness after a sports-related concussion: A randomized clinical trial.

The purpose of this study was to (a) assess the feasibility of recruitment/retention of participants, protocol/resource management, and participant safety, and (b) estimate the size of the effect between the experimental and control groups. This was a feasibility study conducted as a prospective pilot double-blind randomized clinical trial. Subjects aged 10-23 years old with acute concussion and dizziness were enrolled from sports medicine centers. Forty-one participants were randomized into treatment and were seen for physical therapy beginning at 10 days post-concussion. Subjects in the experimental group received individually tailored, pragmatically delivered progressive interventions. Subjects in the control received prescriptive sham to minimally progressive interventions. The two primary outcomes were medical clearance for return-to-play and symptomatic recovery. The median number of days to medical clearance for the experimental group was 15.5 and for the control was 26. The median number of days to symptomatic recovery was 13.5 for the experimental group and was 17 for the control. According to Cox proportional hazards regression for time to medical release for return-to-play, the experimental group demonstrated a hazard ratio of 2.91 (95% CI: 1.01, 8.43) compared to the control. For time-to-symptomatic recovery, those in the experimental group demonstrated a hazard ratio of 1.99 (95% CI: 0.95, 4.15) compared to the control. The results indicate that it is feasible and safe to complete this type of intervention study. The results provide strong support for the allocation of resources to conduct well-powered randomized clinical trials of this intervention.

IFT88 influences chondrocyte actin organization and biomechanics.

OBJECTIVES: Primary cilia are microtubule based organelles which control a variety of signalling pathways important in cartilage development, health and disease. This study examines the role of the intraflagellar transport (IFT) protein, IFT88, in regulating fundamental actin organisation and mechanics in articular chondrocytes. METHODS: The study used an established chondrocyte cell line with and without hypomorphic mutation of IFT88 (IFT88(orpk)). Confocal microscopy was used to quantify F-actin and myosin IIB organisation. Viscoelastic cell and actin cortex mechanics were determined using micropipette aspiration with actin dynamics visualised in live cells transfected with LifeACT-GFP. RESULTS: IFT88(orpk) cells exhibited a significant increase in acto-myosin stress fibre organisation relative to wild-type (WT) cells in monolayer and an altered response to cytochalasin D. Rounded IFT88(orpk) cells cultured in suspension exhibited reduced cortical actin expression with reduced cellular equilibrium modulus. Micropipette aspiration resulted in reduced membrane bleb formation in IFT88(orpk) cells. Following membrane blebbing, IFT88(orpk) cells exhibited slower reformation of the actin cortex. IFT88(orpk) cells showed increased actin deformability and reduced cortical tension confirming that IFT regulates actin cortex mechanics. The reduced cortical tension is also consistent with the reduced bleb formation. CONCLUSIONS: This study demonstrates for the first time that the ciliary protein IFT88 regulates fundamental actin organisation and the stiffness of the actin cortex leading to alterations in cell deformation, mechanical properties and blebbing in an IFT88 chondrocyte cell line. This adds to the growing understanding of the role of primary cilia and IFT in regulating cartilage biology.

Genetic characterization and antimicrobial resistance of Staphylococcus aureus isolated from bovine milk in Tunisia.

Staphylococcus aureus is a major agent of bovine mastitis in dairy herds, causing economic losses in dairy industry worldwide. In addition, milk and milk-products contaminated by Staph. aureus can cause harmful human diseases. The aim of this study was to characterize Staph. aureus strains isolated from dairy farms in Tunisia. Bulk tank milk (n = 32) and individual cow milk (n = 130) samples were collected during the period of 2013-2014. Forty-three Staph. aureus isolates were recovered and typed by spa typing, 16S-23S rRNA intergenic spacer (RS-PCR) and multiplex PCRs for 22 virulence genes. Antimicrobial resistance was also investigated with a disc diffusion test. A selected subsample of 22 strains was additionally genotyped by multilocus sequence typing. Seventeen spa types were recovered, and t2421 (n = 10), t521 (n = 6) and t2112 (n = 5) were the most common. Fourteen different RS-PCR genotypes grouped into 11 clusters were detected in our study, with predominance of the RVI genotype (n = 24). Eight sequence types were identified and Clonal Complex 97, corresponding to RS-PCR cluster R, was the most common (n = 10), followed by CC1 (n = 4), CC15 (n = 3) and other four accounting for one or two strains. Different combinations of virulence genes were reported, and enterotoxin genes were present in few strains (seh, n = 4; sea, n = 2; sea and seh, n = 2; sec and sel, n = 2). The majority of strains were resistant only to penicillin; only one strain was found to be multiresistant and no methicillin-resistant Staph. aureus was demonstrated. Our study reported the isolation of CC97 from bovine milk in Tunisia for the first time and confirmed the relevance of this lineage in intramammary infection in cows. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the characteristics of Staphylococcus aureus isolated from bulk tank and individual cow milk in Tunisia. All strains were genotyped by spa typing and RS-PCR, a method based on the amplification of the 16S-23S rRNA intergenic spacer region, and multiplex PCRs for 22 virulence genes. A selected subsample of strains was also genotyped by multilocus sequence typing. All strains were tested for antimicrobial resistance. Our study evidences a predominance of strains belonging to Clonal Complex 97. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported.

Effect of Tannin and Species Variation on In vitro Digestibility, Gas, and Methane Production of Tropical Browse Plants.

Nineteen tanniferous browse plants were collected from South Africa to investigate their digestibility, gas production (GP) characteristics and methane production. Fresh samples were collected, dried in forced oven, and ground and analyzed for nutrient composition. In vitro GP and in vitro organic matter digestibility (IVOMD) were determined using rumen fluid collected, strained and anaerobically prepared. A semi- automated system was used to measure GP by incubating the sample in a shaking incubator at 39°C. There was significant (p<0.05) variation in chemical composition of studied browses. Crude protein (CP) content of the species ranged from 86.9 to 305.0 g/kg dry matter (DM). The neutral detergent fiber (NDF) ranged from 292.8 to 517.5 g/kg DM while acid detergent fiber (ADF) ranged from 273.3 to 495.1 g/kg DM. The ash, ether extract, non-fibrous carbohydrate, neutral detergent insoluble nitrogen, and acid detergent insoluble nitrogen and CP were negatively correlated with methane production. Methane production was positively correlated with NDF, ADF, cellulose and hemi-cellulose. Tannin decreased GP, IVOMD, total volatile fatty acid and methane production. The observed low methanogenic potential and substantial ammonia generation of some of the browses might be potentially useful as rumen manipulating agents. However, a systematic evaluation is needed to determine optimum levels of supplementation in a mixed diet in order to attain a maximal depressing effect on enteric CH4 production with a minimal detrimental effect on rumen fermentation of poor quality roughage based diet.

Synergistic effects of thermally reduced graphene oxide/zinc oxide composite material on microbial infection for wound healing applications.

Infections originating from pathogenic microorganisms can significantly impede the natural wound-healing process. To address this obstacle, innovative bio-active nanomaterials have been developed to enhance antibacterial capabilities. This study focuses on the preparation of nanocomposites from thermally reduced graphene oxide and zinc oxide (TRGO/ZnO). The hydrothermal method was employed to synthesize these nanocomposites, and their physicochemical properties were comprehensively characterized using X-ray diffraction analysis (XRD), High-resolution transmission electron microscopy (HR-TEM), Fourier-transform infrared (FT-IR), Raman spectroscopy, UV-vis, and field-emission scanning electron microscopy (FE-SEM) techniques. Subsequently, the potential of TRGO/ZnO nanocomposites as bio-active materials against wound infection-causing bacteria, including Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, was evaluated. Furthermore, the investigated samples show disrupted bacterial biofilm formation. A reactive oxygen species (ROS) assay was conducted to investigate the mechanism of nanocomposite inhibition against bacteria and for further in-vivo determination of antimicrobial activity. The MTT assay was performed to ensure the safety and biocompatibility of nanocomposite. The results suggest that TRGO/ZnO nanocomposites have the potential to serve as effective bio-active nanomaterials for combating pathogenic microorganisms present in wounds.

Nutritive Value of Grasses in Semi-arid Rangelands of Ethiopia: Local Experience Based Herbage Preference Evaluation versus Laboratory Analysis.

We examined the nutritive value of common grass species in the semi-arid rangelands of Borana in southern Ethiopia using local experience based herbage preference (LEBHP) perception and laboratory techniques. Local pastoralists in the study area were asked to identify common grass species and rank them according to the species' preferences and palatability to cattle. The pastoralists listed a total of 15 common grass species which were then sampled during the main rain and cold dry seasons and analyzed for crude protein (CP), Neutral Detergent Fiber (NDF), Acid Detergent Fiber (ADF) and ash content to verify pastoralists' claim regarding the quality of individual species. The relative feed value (RFV) and dry matter digestibility (DMD) were also calculated using NDF and ADF contents. Spearman's rank correlation was used to examine possible relationships between laboratory results and pastoralists' experience on grass quality. Cenchrus ciliaris, Chrysopogon aucheri, Digitaria milanjiana, Eragrostis papposa and Panicum maximum were the top five species based on LEBHP perception. There were indications of inconsistency in terms of LEBHP perception among the different pastoral communities. The chemical composition of all grass species showed significant (p<0.05) variation between sites, seasons and species. The results showed that the CP values for the Borana rangelands were in the range of 8.7% in the main rain season to 5.1% for the cold dry season. The fiber constituents were relatively low in the main rain season compared to the cold dry season. Overall, Digitaria milanjiana had the highest CP (16.5%) content, while the least was recorded with Heteropogon contortus (10.8) and Aristida adoensis (9.8%) during the main rain season. It seems that the spatial variability of landscapes within the wider geographical regions, soil properties and texture, and land-use patterns probably contributed to site differences in species quality. Generally, the RFV of individual grass species was significantly (p<0.05) varied between and within sites. The ranking of species by pastoralists according to their preferences by cattle was highly correlated with the chemical composition of laboratory results of individual grass species with 'r' values for CP (0.94), ash (0.95), NDF (-0.98), ADF (-0.93) and ADL (-0.93). We suggest the complimentary use of LEBHP and laboratory techniques in evaluating the nutritive quality of rangeland forage species for sustainable animal production.

Synthesis and numerical simulation of formamidinium-based perovskite solar cells: a predictable device performance at NIS-Egypt.

Formamidinium lead triiodide (δ-FAPbI3)-based perovskite solar cells showed remarkable potential as light harvesters for thin-film photovoltaics. Herein, the mechanochemical synthesis of δ-FAPbI3, MAPbI3, and mixed-cation FA1-xMAxPbI3 with (x = 0.3, 0.5, and 0.7) perovskite materials were prepared as a novel green chemistry method for scaling up production. Crystallinity, phase identification, thermal stability, optoelectronic properties, and nanoscale composition are discussed. The results demonstrated that the prepared mixed-cation samples are enhanced in the visible absorption region and are consistent with previous works. The crystal structure of δ-FAPbI3 was altered to a cubic structure due to the change in FA-cation. Moreover, the performance of [Formula: see text]-FA-based perovskites was investigated using the Solar Cell Capacitance Simulator (SCAPS-1D) software. The validity of the device simulation was confirmed by comparing it to real-world devices. The photovoltaic characteristics and impact of absorber thickness on device performance were explained. The [Formula: see text]-FA-based solar cell with a 50% MA-doped molar ratio shows a better performance with an efficiency of 26.22% compared to 8.43% for δ-FAPbI3. The outcome results of this work confirm the beneficial effect of mixed cations on device operation and advance our knowledge of the numerical optimization of perovskite-based solar cells.

High prevalence of mec complex C and ccrC is independent of SCCmec type V in Staphylococcus haemolyticus.

Staphylococcus haemolyticus is one of the most clinically relevant coagulase-negative staphylococci (CoNS), particularly in immunocompromised patients; however, little is known regarding its molecular epidemiology. In this work, we characterized the genetic background and the SCCmec region of 36 methicillin-resistant S. haemolyticus (MRSHae) and 10 methicillin-susceptible S. haemolyticus (MSSHae) collected from neutropenic patients in Tunisia between 2002 and 2004. The molecular characterization of MRSHae by pulsed-field gel electrophoresis (PFGE) showed that the great majority of the isolates (77.8%) belonged to only four types. SCCmec typing by polymerase chain reaction (PCR) and Southern hybridization showed that isolates belonging to each PFGE type could carry either one or two SCCmec types. SCCmec V was the most common, but mec complex C was frequently associated to ccr allotypes other than ccrC. The mec complex class C was predominant in MRSHae (47%) and ccrC was predominant among both methicillin-resistant and -susceptible isolates (31 and 50%, respectively). Interestingly, one half (50%) of the MRSHae isolates analyzed lacked the known ccr complexes (ccrAB and ccrC), although they carried the mecA. Conversely, all MSSHae carrying a ccrC complex were multidrug-resistant, although they lack the mecA. The results suggest that ccrC and mec complex C are frequent and may exist autonomously and independently of SCCmec type V in S. haemolyticus. Moreover, the data obtained suggest that small chromosomal rearrangements promoting the loss or structural variation of mec and ccr complex appear to occur frequently, which probably provide S. haemolyticus with a specialized means for SCCmec trapping and/or diversification.

Detection and characterization of Shigella species isolated from food and human stool samples in Nabeul, Tunisia, by molecular methods and culture techniques.

AIMS: This study was designed to isolate Shigella spp. strains from food and stool samples by a combination of PCR and culture methods and characterize their serotypes, antibiotic resistance profiles, virulence genes and pulsed-field gel electrophoresis (PFGE) patterns to investigate possible clonal relationships amongst strains circulating. METHODS AND RESULTS: Six Shigella spp. strains were isolated from 280 food samples against 16 Shigella isolates from 236 stool samples of symptomatic patients and asymptomatic food handlers during the period from January 2007 to December 2009 in Public Health Regional Laboratory of Nabeul. The detection of ipaH, ipaBCD, ial, ShET-1 and ShET-2 was performed by a PCR technique with specific primers. CONCLUSIONS: The use of PCR technique improved the rate of detecting Shigella in stool samples from 6·7 to 14% and in food samples from 2·1 to 8·6%. Percentage of Shigella isolates and ipaH-specific PCR demonstrated a marked pattern of seasonality, increasing in summer and fall seasons for human and food isolates. Amongst the environmental strains, 50% of isolates were invasive. However, for the 16 clinical strains isolated, nine were found to be positive for both ial and ipaBCD gene and 11 were found to produce ShET-1 and/or ShET-2. XbaI PFGE analysis revealed the presence of a predominant clone amongst Shigella sonnei strains recovered from different sources circulating in Nabeul, Tunisia, throughout the years 2007-2009. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the existence of Shigella in food samples and dispersion of different virulence genes amongst these isolates, which appear to constitute an environmental source of epidemic spread. The clonal relationships amongst strains isolated from food elements and human stools indicate the incrimination of different kinds of foods as vehicle of transmission of Shigella, which are usually escaped from detection by traditional culture methods.

New fabrication method for di-indium tri-sulfuric (In2S3) thin films.

Di-indium tri-sulfuric (In2S3) thin films are fabricated with annealing indium thin films in a sulfur environment. The effect of both annealing temperature and pressure on the structure, morphology, Raman, and photoluminescence (PL) spectroscopy has been studied. The X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM) of the prepared thin films showed different structural phases and morphology with varying annealing temperature and pressure. Energy dispersive X-ray (EDX) analysis confirmed the chemical composition and the atomic ratio of In/S for the In2S3 thin films. The optimum annealing conditions of In2S3 thin films are 550 °C and 100 Torr. The outcome results revealed a new good growth method for In2S3 thin films to be used for different applications.

Biofilm and siderophore effects on secondary waste water disinfection.

The efficiency of ultraviolet (UV) light disinfection of wastewater effluent using a large-scale pilot system was studied. The relationship between biofilm and siderophore production and UV doses received by Pseudomonas aeruginosa strain ATCC 15442 was determined. UV decreased pyoverdine production and enhanced biofilm production. Consequently external factors conditioned by both pyoverdine and biofilm may affect the UV effect on bacterial disinfection.

Prevalence of resistance phenotypes and genotypes to macrolide, lincosamide and streptogramin antibiotics in Gram-positive cocci isolated in Tunisian Bone Marrow Transplant Center.

To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics in Gram-positive cocci isolated in a Bone Marrow Transplant Center of Tunisia, we tested the antibiotic susceptibility of 172 clinical isolates of Staphylococcus epidermidis, Streptococcus mitis and Enterococcus faecium to macrolide erythromycin and spiramycin, the lincosamide clindamycin and the streptogramin pristinamycin. These three groups of organisms were mostly resistant to macrolides and lincosamide, but were commonly susceptible to pristinamycin. The resistance phenotypes of erythromycin-resistant isolates were determined by the five-disc test with erythromycin, spiramycin, lincomycin, clindamycin and pristinamycin, which showed that most exhibited constitutive MLS resistance. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the prevalence of the erythromycin resistance methylase (erm) (A), erm(B), erm(C), msr(A) and macrolide efflux (mef) (A) genes in the erythromycin-resistant isolates was identified by polymerase chain reaction (PCR) analysis. The resistance was due mainly to the presence of ermB in E. faecium (80%), ermC in S. epidermidis (53%) and mefA in S. mitis (65%).

Evaluation of five commercial real-time PCR assays for detection of Mycoplasma pneumoniae in respiratory tract specimens.

The performances of five commercial TaqMan real-time PCR assays for the detection of Mycoplasma pneumoniae in respiratory tract specimens were evaluated in comparison with an in-house real-time PCR. All kits allowed prompt and specific results, validated by the use of an internal control. The Nanogen kit showed the best clinical sensitivity.

Phenotypic and molecular characterization of beta-lactam resistance and capsular typing of colonizing Haemophilus influenzae strains isolated from neutropenic patients in Tunisia.

OBJECTIVE: The aim of this study was to determine the overall percentage of beta-lactams susceptibility, beta-lactamase production, penicillin binding protein (PBP) modification and serotypes of colonizing Haemophilus influenzae strains. DESIGN: A total of 50 isolates of colonized H. influenzae, isolated from neutropenic patients. The prevalence of beta-lactams resistance and beta-lactamase production were recorded for each strains using E-test strips and chromogenic cephalosporin test, then were determined their resistance genes (bla(TEM) and bla(ROB)) by PCR as well as their capsular types by standard slide agglutination serotyping (SAST) and capsular genes amplification. RESULTS: Thirty-two percent of the 50 strains were amoxicillin resistant, among these, 20% were resistant by beta-lactamase production, and they produced all type TEM beta-lactamase. Four percent of the isolates had PBP modification and three strains (6%) associated the two resistance mechanisms. Slide agglutination serotyping showed that 95.8% of the strains were unencapsulated, and 4.1% were of serogroup b. The result was confirmed by PCR capsular typing. CONCLUSION: By the light of these results, our findings suggest that it becomes important to follow the evolution of the resistance background of our strains, and that the majority of colonizing H. influenzae strains isolated in our center are unencapsulated.

Enterococcus faecium isolated from bone marrow transplant patients in Tunisia: high prevalence of antimicrobial resistance and low pathogenic power.

OBJECTIVES: Investigation of the occurrence and antibiotic susceptibility of Enterococcus faecium isolates, collected during four years from neutropenic patients at the Tunisian bone marrow transplantation centre. MATERIALS AND METHODS: E. faecium strains were identified by conventional methods and by the Api20 Strep (Bio-Mérieux, France). Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton agar and interpreted as recommended by CA-SFM. MICs of ampicillin, vancomycin, and teicoplanin were determined by E-test method. RESULTS: Two hundred and thirty five E. faecium isolates were recovered from stool cultures or rectal swabs (229), throat (three), urine (two), and pus of wound (one). None was responsible for bacteraemia. Ampicillin resistance, without production of beta-lactamase, was observed in 43.8% of isolates. All the isolates were susceptible to glycopeptides. High rates of resistance were observed: high-level resistance (HLR) to gentamicin (33.6%), HLR to kanamycin (55.7%), HLR to streptomycin (47.6%), erythromycin (86.4%), ciprofloxacin (78.7%), rifampicin (85%), and tetracycline (43%). Strains with HLR to gentamicin were significantly more resistant to ampicillin and streptomycin. Multiple drug resistance was observed in most isolates. CONCLUSION: These findings demonstrated the low pathogenic power of E. faecium in our patients, and the high frequencies of resistance to ampicillin and aminoglycosides. In the absence of glycopeptide-resistance, vancomycin remains an alternative treatment against multidrug resistant strains.

[Identification of SHV-type extended spectrum beta-lactamase genes in Pseudomonas aeruginosa by PCR-restriction fragment length polymorphism and insertion site restriction-PCR]. / Identification des gènes de beta-lactamase à spectre étendu de type SHV chez Pseudomonas aeruginosa par PCR-restriction fragment lengthpolymorphism et insertion site restriction-PCR.

We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.

Detection of SHV-1 beta-lactamase in Pseudomonas aeruginosa strains by genetic methods.

Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.