Pathogenic Impacts of Bacillus cereus Strains on Crassostrea gigas.

Regarding the economic importance of bivalve farming, a great deal of interest has recently been devoted to studying the pathogenesis of infectious diseases of these mollusks to prepare for public health emergencies. Bacillus cereus is one of these pathogens; it is a ubiquitous soil bacterium responsible for many types of gastrointestinal diseases associated with food. This study was conducted to determine the pathogenic effect of B. cereus on Crassostrea gigas. This effect was studied by assessing hemocytes death using flow cytometry analysis. The results showed that only ∼15% of C. gigas were able to survive after B. cereus artificial infection with 108 CFU (colony-forming unit)/oyster. Evenly, the percentage of nonviable hemocytes gradually increased with the concentration of B. cereus, with a peak value of ∼40% after infection. Indeed, findings showed that this strain is harmful to C. gigas.

The Synbiotic Effect of Probiotics and Dried Spirulina platensis or Phycocyanin on Biofilm Formation by Salmonella Typhimurium and Staphylococcus aureus.

This study aimed to evaluate the synbiotic effect of probiotics and dried Spirulina platensis or phycocyanin on autoaggregation, coaggregation, and the inhibition of biofilm formation by Salmonella Typhimurium and Staphylococcus aureus on 96-well microtiter plates and Human colon carcinoma cells-116 surfaces. The results showed that the probiotics strains cultured in the presence of S. platensis exhibited the highest autoaggregation values, ranging between 68.5 and 74.2% after 24 h. All probiotic strains with or without S. platensis and phycocyanin showed coaggregation abilities with S. Typhimurium and S. aureus. Interestingly, significant effect of S. platensis and phycocyanin supplementation was observed on the inhibition of the biofilm formation by the selected pathogens during the competition, exclusion, and displacement on abiotic and biotic surfaces.

Atypical Salmonella Typhimurium persistence in the pacific oyster, Crassostrea gigas, and its effect on the variation of gene expression involved in the oyster's immune system.

Salmonella is one of the most important pathogens involved in food intoxication outbreaks, and in many cases, the intoxication has been linked to shellfish which is typically consumed raw. While much is understood about the interactions between Salmonella and vertebrates, much less is known about its relationships with invertebrates, which could be an overlooked and important aspect to better understand the Salmonella interaction with its diversified hosts. The aim of this study was to investigate the effect of preadaptation in seawater microcosms during 12 months on Salmonella Typhimurium by determining its survival capacity within this mollusk over a period of 30 days. The results showed that the stressed bacteria are able to survive in this mollusk at a higher concentration even after thirty days of infection compared to bacteria in the normal state. In order to minimize the effect of an experimental device for one month on the survival of Salmonella, we carried out an in vitro study to determine the number of viable Salmonella in the hemocytes of oysters. Interestingly, we evaluated the effect of the antibacterial activity of different extracts of C. gigas using the solvents (Methanol, Ethanol and acetic acid) specifically against stressed and unstressed Salmonella. Furthermore, we compared the expression of three genes in the oyster Cg-big-def1, timp and sod in response to experimental infections of this mollusk with Vibrio splendidus kb133 and S. Typhimurium LT2DT104 in normal and stressed states. These findings are very important to contribute to explaining several questions about the persistence of S. Typhimurium for a long time in C. gigas and the host's immune response to this microorganism which is considered to be non-virulent for molluscs.

Botanical and Genetic Identification Followed by Investigation of Chemical Composition and Biological Activities on the Scabiosa atropurpurea L. Stem from Tunisian Flora.

Scarce information about the phenolic composition of Scabiosa atropurpurea L. is available, and no carotenoid compounds have been reported thus far. In this study the phenolic and carotenoid composition of this plant was both investigated and associated bioactivities were evaluated. Aiming to obtain extracts and volatile fractions of known medicinal plants to valorize them in the pharmaceutical or food industries, two techniques of extraction and five solvents were used to determine the biologically active compounds. Gas chromatography coupled to flame ionization and mass spectrometry and liquid chromatography coupled to photodiode array and atmospheric pressure chemical ionization/electrospray ionization mass spectrometry highlighted the presence of 15 volatiles, 19 phenolic, and 24 natural pigments in Scabiosa atropurpurea L. stem samples; among them, the most abundant were 1,8-cineole, chlorogenic acid, cynaroside, and lutein. Bioactivity was assessed by a set of in vitro tests checking for antioxidant, antibacterial, antifungal, and allelopathic (against Brassica oleracea L. and Lens culinaris Medik) effects. Scabiosa atropurpurea L. stem extracts presented a considerable antioxidant, antibacterial, and allelopathic potential, with less antifungal effectiveness. These results indicate that the volatile fractions and extracts from S. atropurpurea L. stem could be considered as a good source of bioactive agents, with possible applications in food-related, agriculture, and pharmaceutical fields. Genetic investigations showed 97% of similarity with Scabiosa tschiliensis, also called Japanese Scabiosa.

Microbial Diversity and Pathogenic Properties of Microbiota Associated with Aerobic Vaginitis in Women with Recurrent Pregnancy Loss.

Recurrent pregnancy loss (RPL) is a major reproductive problem that affects approximately 5% of couples. The objective of this study was to assess vaginal flora dysbiosis in women suffering from unexplained RPL and to investigate the pathogenic properties of the microbiota associated with aerobic vaginitis (AV). The study included one hundred fifteen women, 65 with RPL and 50 controls. The diversity of vaginal microbiota isolated was evaluated by molecular sequencing. Then, pathogenic factors, such as acid-resistance, antibiotics susceptibility, and biofilm formation were evaluated. The prevalence of AV was five-fold higher in the RPL group than in the controls (64.6% vs. 12.0%). The most prevalent isolates in the case group were Enterococcus spp. (52%) and Staphylococcus spp. (26%). All bacterial strains tolerate low pH. The prevalence of multidrug resistance (MDR) among all bacteria was 47.7%. Of all strains, 91.0% were biofilm producers. The presence of MDR was found to be related to biofilm formation. The results provide evidence supporting an increased presence of dysbiosis of the vaginal flora, especially AV, in women with RPL in Tunisia. The viability of the AV-associated bacteria and their persistence in the genitals may be due to their ability to resist low pH and to produce a biofilm.

Protective effects of dietary Kefir against aflatoxin B1-induced hepatotoxicity in Nile tilapia fish, Oreochromis niloticus.

The effect of dietary Kefir supplementation on the biometric, biochemical, and histological parameters of Nile tilapia (Oreochromis niloticus) exposed to aflatoxin B1 (AFB1, 200 µg/kg diet) contamination was studied. The yeasts were dominant in Kefir followed by lactic and acetic acid bacteria. The Kefir showed relatively interesting antioxidant potential in the DPPH• (IC50 = 0.9 ± 0.02 mg/ml) and ABTS•+ (IC50 = 2.2 ± 0.03 mg/ml) scavenging activities, Fe3+-reducing power (EC0.5 = 1.2 ± 0.01 mg/ml), and ß-carotene bleaching assay (IC50 = 3.3 ± 0.02 mg/ml). Three hundred and sixty Nile tilapia weighing 23 ± 5 g were divided into four groups (30 fish/group with 3 replicates), and fed with diets containing Kefir (D2), AFB1 (D3), and Kefir+AFB1 (D4) for 4 weeks, whereas D1 was kept as control group where fish were fed with basal diet. The Kefir supplementation in D4 group significantly increased (p < .05) the percent weight gain as compared to D3 group. Moreover, Kefir improved the antioxidant enzymes in the liver, such as catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) activities, that significantly increased (p < .05) by 2-, 3-, and 1.5-folds, respectively, as compared to D3 group. The Kefir treatment significantly decreased (p < .05) the liver malonaldehyde content by ~50% as compared to D3 group. Histopathological analysis revealed the hepatoprotective effects of Kefir by showing normal liver histological architecture in D4 group, as compared to degenerative changes observed in D3 group. These results suggest that Kefir could be considered as a potential probiotic in Nile tilapia feed to mitigate the AFB1 harmful effects.

Does probiotic Kefir reduce dyslipidemia, hematological disorders and oxidative stress induced by zearalenone toxicity in wistar rats?

Zearalenone (ZEA) is a toxic metabolite of the genus Fusarium, which causes hepatotoxicity and induces oxidative stress. Kefir is an important probiotic dairy-product showing important in vitro antioxidant potential. In this study, the effect of Kefir supplementation to mitigate ZEA toxicity in rats was investigated. Animals were divided into four groups of five rats each, which received sterile milk (200 µL/day) during the first week. Then, they were switched to Kefir (200 µL/day), ZEA (40 mg/kg b. w./day) and Kefir + ZEA for the second week. Hematological and biochemical parameters, as well as liver histological analysis were determined. Kefir administration prevented the changes occurred in the count of all blood cells, and improved the antioxidant enzymes in the liver, such as catalase, glutathione peroxidase and superoxide dismutase activities that increased by 6, 4.5 and 1.3 folds, respectively, compared to ZEA group. Interestingly, the concurrent regimen Kefir + ZEA removed ZEA residues in the serum and liver. Furthermore, the Kefir + ZEA group showed a reduction in the levels of bilirubin, alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase and hepatic malonaldehyde by ∼82, 54, 66, 50 and 36%, respectively, compared to the ZEA group. The histopathological analysis showed a normal liver histological architecture in Kefir + ZEA group, while degenerative changes were observed in ZEA group. These results suggest that Kefir as probiotic consortium may have a hepatoprotective effect against ZEA poisoning.

Control of Staphylococcus aureus methicillin resistant isolated from auricular infections using aqueous and methanolic extracts of Ephedra alata.

In the current study the potential use of aqueous and methanolic extracts of Ephedra alata aerial parts as biological control agent against pathogenic bacteria and especially Staphylococcus aureus methicillin resistant isolated from auricular infections was evaluated. Chemical tests and spectrophotometric methods were used for screening and quantification of phytochemicals. The assessment of the antioxidant activity was accomplished by DPPH and ABTS radicals scavenging assays. Extracts were evaluated for their antibacterial efficacy by diffusion and microdilution methods. Biofilm inhibition was tested using XTT assay and the cytotoxicity of extracts was carried out on Vero cell line. The GC-FID analysis revealed that E. alata was rich in unsatured fatty acids. In addition, the aqueous extract had the highest flavonoid and protein contents (30.82 mg QE /g dry extract and 98.92 mg BSAE/g dry extract respectively). However, the methanolic extract had the highest phenolic, sugars and tannins. The antioxidant activity demonstrated that the aqueous extract exhibited the strong potency (IC50 ranged between 0.001 and 0.002 mg/mL). Both extracts displayed antimicrobial activity on Gram negative and positive strains. They were effective against S. aureus isolated from auricular infections. The tested extracts were able to inhibit biofilm formation with concentration-dependent manner. Moreover, no cytotoxic effect on Vero cells line was demonstrated for the extracts. Overall, our findings highlight the potential use of E. alata extract as a novel source of bioactive molecules with antioxidant, antibacterial and antiobiofilm effects for the control of infectious disease especially those associated to S. aureus methicillin resistant.

Decolorization and phytotoxicity reduction of reactive blue 40 dye in real textile wastewater by active consortium: Anaerobic/aerobic algal-bacterial-probiotic bioreactor.

The textile dyeing and printing industries has led to extensive environmental pollution and severely threatens ecosystems. The best microbial species for such application was selected among the isolated bacterial populations by conducting CI Reactive Blue 40 (CI RB 40) batch degradation studies with the bacterial-algal-probiotic strains. In this study, three suitable species (Pseudomonas putida, Chlorella and Lactobacillus plantarum) were applied to degrade and detoxify CI RB 40, a reactive diazo dye in Real Textile Wastewater, used in textile dyeing industry worldwide. Process parameters were optimized using Response Surface Methodology and under the optimum conditions (e.g., inoculum size of 10%), temperature of 35 °C, 150 ppm, and time of 6 days). The maximum COD and color removal efficiencies, when tested with 1000 ppm of dye using batch reactors were found to be 89% and 99%, respectively. Our results showed also that bacteria had a high decolorization capacity. The regression analysis revealed a good match of the experimental data to the second-order polynomial with a high coefficient of determination (R2). UV-Visible and FTIR spectroscopy analysis confirmed the biodegradation of CI RB 40. Finally, toxicity of CIRB 40 before and after biodegradation was studied and the detoxification of CIRB 40 dye solution after biodegradation process was confirmed.

Legionella pneumophila sg1-sensing signal enhancement using a novel electrochemical immunosensor in dynamic detection mode.

This work presents a comparison between static and dynamic modes of biosensing using a novel microfluidic assay for continuous and quantitative detection of Legionella pneumophila sg1 in artificial water samples. A self-assembled monolayer of 16-amino-1-hexadecanethiol (16-AHT) was covalently linked to a gold substrate, and the resulting modified surface was used to immobilize an anti-Legionella pneumophila monoclonal antibody (mAb). The modified surfaces formed during the biosensor functionalization steps were characterized using electrochemical measurements and microscopic imaging techniques. Under static conditions, the biosensor exhibited a wide linear response range from 10 to 108 CFU/mL and a detection limit of 10 CFU/mL. Using a microfluidic system, the biosensor responses exhibited a linear relationship for low bacterial concentrations ranging from 10 to 103 CFU/mL under dynamic conditions and an enhancement of sensing signals by a factor of 4.5 compared to the sensing signals obtained under static conditions with the same biosensor for the detection of Legionella cells in artificially contaminated samples.

Dietary administration effects of exopolysaccharide from potential probiotic strains on immune and antioxidant status and nutritional value of European sea bass (Dicentrarchus labrax L.).

The use of biological immunostimulants is considered a valuable practice to improve culture conditions in aquaculture sector that may help to increase production and maintain healthy environment. We undertook this study in order to evaluate the potential effect of the administration of two exopolysaccharides (EPS) "EPLB" and "EPB" derived from potential probiotic strains on immune and antioxidant status of European sea bass (Dicentrarchus labrax L.) larvae. In order to find out if the EPSs have an effect on the biochemical composition during the trial period, the nutritional value has been evaluated. The results revealed that expression levels of immune-relevant genes (infg, Il1b, Il8, Il6 and tcr-ß) in the gut and head kidney and the scavenging enzymes (cat, sod, gr) genes in the liver were modulated. In fact, the dietary supplementation with the tested EPSs, significantly enhances the expression of immune-associated genes in the head-kidney, particularly infg and tcrß, as well as catalase gene in liver. During the period of study, EPSs administration did not affect the fatty acid profiles of larvae, which is balanced. This is confirmed by the Docosahexaenoic acid / Eicosapentaenoic acid ratio and demonstrates that EPLB and EPB can be administrated without any negative effect on biochemical composition of European sea bass. The present findings provided evidence that the tested EPSs with antibacterial and antioxidant activities can enhance immune response without negative effect on the biochemical composition. The used EPSs can be considered as a good source of natural functional aquafeed ingredients for European sea bass.

Fermented Seeds ("Zgougou") from Aleppo Pine as a Novel Source of Potentially Probiotic Lactic Acid Bacteria.

Microorganisms inhabiting fermented foods represent the main link between the consumption of this food and human health. Although some fermented food is a reservoir of potentially probiotic microorganisms, several foods are still unexplored. This study aimed at characterizing the probiotic potential of lactic acid bacteria isolated from zgougou, a fermented matrix consisting of a watery mixture of Aleppo pine's seeds. In vitro methods were used to characterize the safety, survival ability in typical conditions of the gastrointestinal tract, and adherence capacity to surfaces, antimicrobial, and antioxidant activities. Strains belonged to the Lactobacillus plantarum group and Enterococcus faecalis showed no DNase, hemolytic, and gelatinase activities. In addition, their susceptibility to most of the tested antibiotics, satisfied some of the safety prerequisites for their potential use as probiotics. All the strains tolerated low pH, gastrointestinal enzymes, and bile salts. They displayed a good antibacterial activity and antibiofilm formation against 10 reference bacterial pathogens, especially when used as a cell-free supernatant. Furthermore, the lactic acid bacteria (LAB) strains inhibited the growth of Aspergillus flavus and Aspergillus carbonarius. Finally, they had good antioxidant activity, although depending on the strain. Overall, the results of this work highlight that zgougou represents an important reservoir of potentially probiotic LAB. Obviously, future studies should be addressed to confirm the health benefits of the LAB strains.

Potential Use of Probiotic Consortium Isolated from Kefir for Textile Azo Dye Decolorization.

Azo dyes are recalcitrant pollutants, which are toxic, carcinogenic, mutagenic and teratogenic, that constitute a significant burden to the environment. The decolorization and the mineralization efficiency of Remazol Brillant Orange 3R (RBO 3R) was studied using a probiotic consortium (Lactobacillus acidophilus and Lactobacillus plantarum). Biodegradation of RBO 3R (750 ppm) was investigated under shaking condition in Mineral Salt Medium (MSM) solution at pH 11.5 and temperature 25°C. The bio-decolorization process was further confirmed by FTIR and UV-Vis analysis. Under optimal conditions, the bacterial consortium was able to decolorize the dye completely (>99%) within 12 h. The color removal was 99.37% at 750 ppm. Muliplex PCR technique was used to detect the Lactobacillus genes. Using phytotoxicity, cytotoxicity, mutagenicity and biototoxicity endpoints, toxicological studies of RBO 3R before and after biodegradation were examined. A toxicity assay signaled that biodegradation led to detoxification of RBO 3R dye.

[Bacteriologic features of urinary tract infections in children in the Sousse area, Tunisia]. / Particularités bactériologiques des infections urinaires chez l'enfant dans la région de Sousse, Tunisie.

Urinary tract infection (UTI) is frequent in childhood. Our purpose is to determine the bacteriologic profile of UTI in children through a retrospective study of 1281 urinary specimens analysed in the Laboratory of Microbiology of F. Hached University hospital of Sousse between 1997 and 2002 (2000 except). The most frequent pathogens recovered were E. coli (71%), K. pneumoniae (10%) P. mirabilis (8%), Staphylococcus (1.6%), P. aeruginosa (1%) and others (2%). E. coli susceptibility to antibiotics was characterised by the high resistance percentage to amoxicillin (60%), to amoxicilline - acid clavulanic (54%) and to cotrimoxazole (40%). The resistance percentage to third generation cephalosporins, to aminoglycosides and to nitrofurane remained very low, respectively of 1.5%, 1% and 1%. High resistance rates among K. pneumoniae strains towards to amoxicillin - acid clavulanic and Cefotaxim, respectively of 63 and 39% were noticed. The resistance percentages to amikacin and cotrimoxazole were respectively of 17 and 65%, but only of 4% to nitrofurane. 70% of P. mirabilis strains were resistant to amoxicillin, 63% of them remained susceptible to amoxicillin - acid clavulanic. No resistance was shown to amikacin against 31% towards cotrimoxazole.

AFM, CLSM and EIS characterization of the immobilization of antibodies on indium-tin oxide electrode and their capture of Legionella pneumophila.

The microscopic surface molecular structures and properties of monoclonal anti-Legionella pneumophila antibodies on an indium-tin oxide (ITO) electrode surface were studied to elaborate an electrochemical immunosensor for Legionella pneumophila detection. A monoclonal anti-Legionella pneumophila antibody (MAb) has been immobilized onto an ITO electrode via covalent chemical bonds between antibodies amino-group and the ring of (3-Glycidoxypropyl) trimethoxysilane (GPTMS). The functionalization of the immunosensor was characterized by atomic force microscopy (AFM), water contact angle measurement, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) in the presence of [Fe(CN)6](3-/4-) as a redox probe. Specific binding of Legionella pneumophila sgp 1 cells onto the antibody-modified ITO electrode was shown by confocal laser scanning microscopy (CLSM) imaging and EIS. AFM images evidenced the dense and relatively homogeneous morphology on the ITO surface. The formation of the complex epoxysilane-antibodies acting as barriers for the electron transfer between the electrode surface and the redox species in the solution induced a significant increase in the charge transfer resistance (Rct) compared to all the electric elements. A linear relationship between the change in charge transfer resistance (ΔRct=Rct after immunoreactions - Rct control) and the logarithmic concentration value of L. pneumophila was observed in the range of 5 × 10(1)-5 × 10(4) CFU mL(-1) with a limit of detection 5 × 10(1)CFU mL(-1). The present study has demonstrated the successful deposition of an anti-L. pneumophila antibodies on an indium-tin oxide surface, opening its subsequent use as immuno-captor for the specific detection of L. pneumophila in environmental samples.

Etiology of Acute Diarrhea in Tunisian Children with Emphasis on Diarrheagenic Escherichia coli: Prevalence and Identification of E. coli Virulence Markers.

BACKGROUND: Diarrheal diseases can be caused by viral, bacterial and parasitic infections. This paper provides a preliminary image of diarrhea with regards to etiology and epidemiologic factors in Tunisian children less than five years of age. METHODS: Overall, 124 diarrhoeal stools were collected from patients suffering from acute diarrhea and 54 stool samples from healthy children. All stools were examined for the presence of enteric pathogens. RESULTS: In diarrheagenic children, 107 pathogenic bacteria were isolated (12 Salmonella spp. (9.7%) and 95 diarrheagenic Escherichia coli strains (76.6%): 29 enteroaggregative E.coli (EAEC) (23.4%), 15 enteroinvasive E.coli (EIEC) (12.1%), 17 enteropathogenic E.coli (EPEC) (13.7%), 26 enterotoxigenic E.coli (ETEC) (21%) and 2 enterohemoragic E.coli (EHEC) (1.6%). However, in the control group, 23 pathogenic E.coli strains were isolated (42.6%): 8 EAEC (14.8%), 12 EIEC (22.2%) and 3 EPEC (5.5%). Among diarrheagenic E.coli (DEC), only ETEC strains were significantly recovered from diarrheagenic children than from healthy controls (P < 0.0003). Group A rotavirus was identified in 33.9% (n=42) of diarrheagenic children and in 11.1% among the control group (n=6). Concerning norovirus, 8.9% (n=11) of the samples collected from diarrheagenic children and 9.2% (n=5) from the control group were positive. The prevalence of rotaviruses and Salmonella spp were also significantly higher in patients with diarrhea than in controls (P = 0.002 and P < 0.019, respectively). Finally, enteropathogenic parasites (Entamoeba coli and cryptosporidium Oocystes) were isolated from 4.8% and 9.2% of diarrheagenic and control children, respectively. CONCLUSION: These results provide baseline data about the relative importance of different enteropathogens in Tunisian children.

Investigation of the clonal dissemination of Klebsiella pneumoniae isolates producing extended-spectrum beta-lactamases in a neonatal ward, Sousse, Tunisia.

This study was designed to investigate the spread of extended-spectrum beta-lactamase producing Klebsiella pneumoniae (ESBL-Kp) strains in Sousse hospital, during 7-month period by using phenotypic and genotypic markers. A total of 57 clinical isolates of ESBL-Kp, 22 strains recovered from seriously infected neonates and 35 strains recovered from colonized neonates and hospitalized in the neonatal ward of Sousse hospital, Tunisia, was subjected to 99 carbon source utilization tests, ribotyping and pulse-field gel electrophoresis (PFGE) profiles of total genomic DNA. Biotyping, ribotyping and PFGE typing showed that four different clones circulated in the neonatal ward between January and July 1997 and suggested that the epidemic strain belonged to the same biotype, ribotype and PFGE pattern, and was represented by 18 isolates from infected neonates and 28 isolates from colonized neonates. Biotyping, ribotyping and PFGE typing appeared to be reliable methods for distinguishing K. pneumoniae strains. Biotyping, which has the advantage of simplicity and rapidity, may be used as a first screening method.

Nosocomial outbreak caused by Salmonella enterica serotype Livingstone producing CTX-M-27 extended-spectrum beta-lactamase in a neonatal unit in Sousse, Tunisia.

In this study, we report an outbreak of Salmonella enterica serotype Livingstone resistant to extended-spectrum cephalosporins that occurred in a neonatal ward of the maternity department of Farhat Hached Hospital, Sousse, Tunisia, in 2002. A total of 16 isolates were recovered from 16 babies hospitalized in the ward during the period 1 to 16 July. All these babies developed diarrhea, and three of them developed septicemia. All the isolates demonstrated resistance to ceftriaxone and ceftazidime due to the production of an extended-spectrum beta-lactamase (ESBL). The isolates were also resistant to aminoglycosides (kanamycin, tobramycin, netilmicin, gentamicin, and amikacin) and sulfamethoxazole-trimethoprim. DNA profiles were determined by pulsed-field gel electrophoresis using the XbaI and SpeI endonucleases and by ribotyping with PstI digestion. They yielded the same patterns, showing that the outbreak was caused by a single clone. The ESBL was identified as CTX-M-27 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 40-kb conjugative plasmid. The mobile insertion sequence ISEcp1 was found to be located upstream of bla(CTX-M-27) in the same position as that known for a bla(CTX-M-14) sequence. A new gene named dfrA21, encoding resistance to trimethoprim and carried by a 90-kb plasmid, was characterized. The dfrA21 gene was inserted as a single resistance cassette in a class I integron. The babies were treated with colistin, and all except two recovered. The outbreak came to an end when appropriate actions were taken: patient isolation, hand washing, and disinfection of the ward.