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Previous studies have suggested the use of probiotics, as alternative to antibiotics, to enhance broiler performance. The administration of probiotics in feed has been widely explored; however, few studies have evaluated the in ovo inoculation of probiotics. Therefore, the objective was to evaluate the impact of in ovo inoculation of different concentrations of GalliPro Hatch (GH), an Enterococcus faecium-based probiotic, on hatchability, live performance, and gastrointestinal parameters. Ross x Ross 708 fertile eggs were incubated, and on day 18, injected with the following treatments: 1) 50 μL of Marek's vaccine (MV), 2) MV and 1.4 × 105 cfu GH/50 μL, 3) MV and 1.4 × 106 cfu GH/50 μL, 4) MV and 1.4 × 107 cfu GH/50 μL. On the day of hatch, chicks were weighed, feather sexed, and hatch residue was analyzed. Male birds (640) were randomly assigned to 40 floor pens. On day 0, 7, 14, and 21 of the grow-out phase, performance data were collected. One bird from each pen was used to obtain yolk weight and intestinal segment weight and length. Hatchability was not impacted by any GH treatment (P = 0.58). On day 0, yolk weight was lower for all treatments than for MV alone. On day 0 to 7, feed intake was lower for 105 and 107 GH; the feed conversion ratio (FCR) was lower for all treatments than for MV alone (P = 0.05; P = 0.01, respectively). From day 14 to 21, the 107 GH treatment had higher BW gain (P = 0.05). For day 0 to 21, 107 GH had a lower FCR than MV alone (P = 0.03). On day 0, all GH treatments resulted in heavier tissues and longer jejunum, ileum, and ceca lengths than MV alone (P < 0.05). Spleen weight was higher for 105 and 107 GH than for MV alone. In conclusion, GH does not impact hatchability, and some concentrations improved live performance through the first 21 d of the grow-out phase. These improvements could result from the increased yolk absorption and improved intestinal and spleen morphology seen in this study.

Dietary supplementation with spore-forming Bacillus-based probiotics represents an efficient means to improve gut health while maintaining good broiler performance. This study investigated the potential of two probiotic products in chickens subjected to optimal (Experiment 1) and Clostridium perfringens-challenged (Experiment 2) conditions. The treatments in Experiment 1 were as follows: (i) CON (no probiotic additive), (ii) One-strain Pro (supplemented with Bacillus licheniformis) or (iii) Multi-strain Pro (supplemented with a multistrain Bacillus-based probiotic). The treatment groups in Experiment 2 received the same diets as those in Experiment 1 but were subjected to C. perfringens challenge. Both experiments lasted 35 days. Both products marginally affected broiler performance in the optimal or challenge conditions. In Experiment 1, Multi-strain Pro upregulated the mRNA expression level of 11 out of 15 selected genes, whereas in Experiment 2, this was less evident, and One-strain Pro was more effective. The multistrain probiotic was effective in maintaining gut morphostructure indices and increasing gut wall thickness, which was particularly evident in challenged birds. Neither additive induced bacterial activity (assessed by measuring enzymatic activity and short-chain fatty acid production) in the cecum, and Multi-strain Pro maintained the cecal butyrate concentration in challenged birds as in the challenged CON treatment, in which butyrate concentration was significantly higher than in the One-strain Pro treatment. Our findings indicated that the activity of these single- and multistrain probiotic products varies depending on rearing conditions, and the effect is highly strain- and product-specific. However, the multistrain probiotic apparently had more beneficial effects than the one-strain probiotic in the maintenance of gut functional status under optimal and challenge conditions.

The application of probiotics in broiler feed, to alleviate performance deficiencies due to mild infections by coccidia and Clostridium perfringens, is of increasing interest for the poultry industry. Therefore, our objective was to evaluate the capacity of 3 Bacillus strains and their combination as probiotics in vitro and in vivo. Thus, protein and carbohydrate degradation and C. perfringens growth inhibition capabilities were assessed by colometry measurement and an agar diffusion bioassay, respectively. A total of 2,250 1-day-old male broiler chicks were assigned to 5 dietary treatments: 1) non–probiotic-supplemented control (control); 2) control + DSM 32324 at 0.8 × 106 cfu/g of feed; 3) control + DSM 32325 at 0.5 × 106 cfu/g of feed; 4) control + DSM 25840 at 0.3 × 106 cfu/g of feed; and 5) control + DSM 32324 + DSM 32325 + DSM 25840 at 1.6 × 106 cfu/g of feed. A pathogenic field strain of C. perfringens was used to induce the necrotic enteritis challenge on day 19, 20, and 21. All birds and remaining feed were weighed on pen basis on day 0, 21, 35, and 42, to calculate BW gain and mortality-adjusted feed conversion. Mortality and mortality due to necrotic enteritis were recorded daily. On day 21, 45 birds per treatment were evaluated for macroscopic intestinal necrotic enteritis lesions. Performance data were statistically analyzed using an ANOVA and subjected to a least significant difference comparison. Necrotic enteritis lesion scores were statistically analyzed using nonparametric Kruskal-Wallis test. Dunn's test was used for treatment comparison. The tested strains showed different abilities of degrading protein and carbohydrates and inhibiting C. perfringens growth in vitro. The birds fed the multi-train combination presented significantly better performance and lower necrotic enteritis lesion score than those in the control group. Dietary supplementation with probiotics resulted in significantly lower necrotic enteritis mortality. The results demonstrate the suitability of the evaluated Bacillus multistrain combination as an effective probiotic in C. perfringens–challenged chickens.

Concerns about antibiotic-resistant bacteria and their presence in animal products grow and thus alternatives to use of antibiotics in animal production are being investigated. Probiotics have gained increased focus due to improvements in performance, immune health and pathogen reduction when provided to poultry through feed. These traits may be further improved if probiotics can be provided to the embryo before hatch, before meeting environmental pathogens. The objective was to determine the faith of a probiotic Enterococcus faecium (M74) strain in the yolk sac and intestinal tract of broiler chickens after injection into hatching eggs. E. faecium M74 (1.4 × 107 CFU/egg) was applied in ovo at day 18 of incubation. From 1- and 7-day-old chickens, 20 samples from yolk sac, caecal tonsils and rest of the intestinal tract were subjected to CFU counting. Isolates from a sample subset were typed by pulsed-field gel electrophoresis (PFGE). Enterococci were found in varying numbers: 1.0 × 104–2.2 × 1010 CFU/g. The prevalence of M74 PFGE profiles was high in 1-day-old (88%) and 7-day-old chickens (67%). This demonstrates that the embryos ingested M74 before hatching, that M74 is viable for intestinal colonization through in ovo administration, and that the strain multiplies in the chickens gastrointestinal tract post hatching.

The present study evaluated the impacts of Bacillus subtilis (BAS) inclusion in broiler diets with standard nutrient content or nutrient deficiency (ND) on growth performance (GP) and nutrient digestibility. The 42 d experiment consisted of 6 experimental diets, a diet with standard nutrient content, and 2 diets with different levels of energy and protein deficiency, without or with BAS. At the end of experiment, apparent ileal digestibility coefficients (AIDC) of starch, crude protein (CP), and gross energy (GE) were determined. Furthermore, impacts of BAS supplementation in standard diets on gut histomorphology, bacterial metabolic activity, and composition were evaluated. Performance and AIDC data were subjected to ANOVA using GLM procedure with a 3 (nutrient levels) × 2 (BAS presence/absences) factorial arrangement of treatments. Gut histomorphology and microbiology data, obtained from broilers fed standard diets without (S) and with BAS (SB), were assessed by an independent Student's t-test. The ND in diets was effective enough to cause nutritional stress and negatively affect performance. Inclusion of BAS in both types of diet improved GP, which was due to the fact that adding BAS in these diets led to improvements in AIDC of CP, starch, and GE (P ≤ 0.05). Comparing only 2 experimental groups, S and SB, revealed no impact on bacterial composition and metabolism in the ileum and cecum, except a reduction in ileal lactobacilli number for SB group. Adding BAS to standard diet reduced crypt depth (CD) and increased villus length to CD ratio in the duodenum, whereas it had no impact on other histomorphological variables in the duodenum, jejunum, and ileum. In conclusion, supplementation of broiler diets with probiotic BAS can positively affect growth performance and nutrient digestibility and this positive impact might even be more pronounced in nutrient-deficient diets. However, the extent of the alleviating ability of BAS in nutrient-deficient diets as well as the biological mechanisms for such a phenomenon needs to be studied further.

Endophytic microbes have attracted considerable attention as a completely new source of pharmaceuticals in recent years. An endophytic Fusarium tricinctum was isolated from Rhododendron tomentosum, a shrub that is found widely across the northern hemisphere. The endophytic F. tricinctum produced antimicrobial compounds that were active against Staphylococcus carnosus, Candida albicans and C. utilis. The transcriptome of the F. tricinctum was sequenced, and a total of 12,006 contigs were assembled, having an N50 value of 1390 bp. Analysis of the transcriptomic PD library of F. tricinctum yielded an antimicrobial peptide named Trtesin. The expression of Trtesin transcripts was >1000 fold in the mRNA library originating from PDB-grown fungi exhibiting high antimicrobial activity compared to FG4-grown fungi with no antimicrobial activity. Trtesin was cloned, expressed, and purified in pET32(a), and it consisted of 52 amino acids with 6 cysteine molecules. The molecular weight of Trtesin is 6138.92 Da. An additional N-terminal sequencing was done to confirm the intact peptide, as well as to check the correct amino acid sequence. The MIC of Trtesin was determined against several bacteria as 64 μg/mL, and peptide showed a mild activity against F. oxysporum in agar diffusion assay, as a zone of inhibition of 10 mm was formed at 100 μg of peptide.

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

Objectives Commercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments. Methods cDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains. Results Lucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L. Conclusions The TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.

The aim of the study was to investigate the potential spread of gentamicin resistant (GENR) Escherichia coli isolates or GENR determinants from a Danish university hospital to the waste water environment. Waste water samples were collected monthly from the outlets of the hospital bed wards and the inlet of the related waste water treatment plant (WWTP) from October 2002 to August 2003. Waste water samples were also collected monthly from a residential area in the same period to be able to compare the prevalence of GENRE. coli isolates from hospital related and residential waste water. The waste water isolates were compared to GENRE. coli isolates obtained consecutively from September 2002 to September 2003 from patients mainly with urinary tract infections at the hospital with respect to Pulsed Field Gel Electrophoresis (PFGE) profiles. All isolates were investigated for GENR mechanisms (aac(3)-II, aac(3)-IV, ant(2″)-I, armA), phenotypic resistance pattern, and virulence genes (hlyA, chuA, sfaS, fogG, malX, traT, iutA, fyuA, iroN, cnf1) to investigate if the hospital and waste water could be reservoirs of antimicrobial resistance and virulence. The ability for GENR determinants to transfer horizontally was investigated by mating experiments.A total of 38, 15, 21, and two GENRE. coli were isolated from patients, the hospital outlets, the inlet of the WWTP, and the residential area, respectively. GENRE. coli were more prevalent in waste water from the hospital and the WWTP than in waste water from the residential area. PFGE profiling revealed no spread of specific patient isolates to the waste water. The aac(3)-II gene was detected both in patient and waste water isolates. Furthermore horizontal transfer of the aac(3)-II gene of patient origin to a recipient was shown in vitro, indicating a potential spread of the gene from patient isolates to waste water isolates. Regardless of origin, most isolates exhibited multi-resistance and contained several virulence genes.In conclusion, our study showed a possible spread of aac(3)-II from the hospital to the waste water. Most of the GENRE. coli isolates from both patients and waste water had a multi-resistant phenotype and contained virulence genes and should therefore be considered reservoirs of antimicrobial resistance and virulence genes. Keywords: Escherichia coli, Gentamicin, Dissemination, Antimicrobial resistance, Virulence

Escherichia coli isolates obtained from faeces (n=85) and blood (n=123) were susceptibility tested against 17 antimicrobial agents and the presence of 9 virulence genes was determined by PCR. Positive associations between several antimicrobial resistances and 2 VF genes (iutA and traT) were found among blood isolates, sometimes among faecal isolates.

OBJECTIVES To test the bactericidal activity of human beta-defensins (hBDs) 2 and 3 against extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains. METHODS Thirty-six Klebsiella pneumoniae and seventeen Klebsiella oxytoca ESBL-producing isolates from nosocomial infections were tested. The bactericidal activity of recombinantly synthesized hBD-2 and -3 was tested and the results were given either as lethal doses killing > or = 90% of bacteria (LD90s) or as MBCs (> or = 99.9% killing). RESULTS Except for one intermediately susceptible strain (MBC = 25 mg/L), all other ESBL-producing strains were highly susceptible to both defensins (LD90s and MBCs < or = 12.5 mg/L). CONCLUSIONS The results underline the high efficacy of hBD-2 and -3 against ESBL-producing Klebsiella, making both defensins attractive candidates as antimicrobial agents to combat these increasingly troublesome bacteria.

The presence of tetracycline resistance (Tc r ) genes and class I integrons (in-1), and their ability to cotransfer were investigated in Tc r gram-negative (185 strains) and gram-positive (72 strains) bacteria from Danish farmland and pigsties. The isolates belonged to the groups or species Escherichia coli , Enterobacter spp., Arthrobacter spp., Alcaligenes spp., Pseudomonas spp., and Corynebacterium glutamicum. The 257 isolates were screened for in-1. Eighty-one of the gram-negative isolates were also screened for the Tc r genes tet (A), tet (B), and tet (C), and all ( n = 72) gram-positive isolates were screened for tet (33). Fourteen (7%) of the soil isolates and eleven (25%) of the pigsty isolates contained in-1. All isolates that contained tet genes also contained in-1, except one gram-negative isolate from a pigsty that contained tet (B). All gram-positive isolates with in-1 also contained tet (33). No isolates contained more than one tet gene. The in-1-positive isolates were tested for resistance to selected antimicrobial agents and showed resistance to three to nine drugs. Filter-mating experiments showed cotransfer of Tc r and class I integrons from soil isolates to Escherichia coli and/or Pseudomonas putida . We conclude that soil bacteria in close contact to manure or pigsty environment may thus have an important role in horizontal spread of resistance. Use of tetracyclines in food animal production may increase not only Tc r but also multidrug resistance (caused by the presence tet genes and in-1) in bacteria.

SUMMARYFifty-sevenSalmonellaTyphimurium strains isolated from poultry, swine and animal feed in Poland during the years 1979–1998 and 2000–2002 were analysed with conventional and molecular techniques. Antimicrobial resistance as well as multiresistance was found, respectively, in 80·1% and 56·1% of the isolates and most frequently among isolates from 2000–2002. Of several phage types noted, DT104 was prevalent among poultry, swine and feed isolates. DT104, U302 and non-typable strains had a multiple resistant profile (ACSSuT) due to the presence of class I integrons. Pulse-field gel electrophoresis ofXbaI andBlnI digest showed high genomic similarity between the strains and confirmed clonal spread ofS. Typhimurium infections. Plasmid profiling allowed further differentiation of the strains. We have, therefore, confirmed the appearance ofS. Typhimurium DT104 showing genome integrated integron-mediated antimicrobial resistance in Poland. These findings are significant for public and animal health risks and document the dissemination of DT104 epidemic strains into new geographical regions.

Objectives To identify and characterize the aetiology of an outbreak of extra-intestinal multidrug-resistant Escherichia coli infections in elderly patients in Israel. Methods Extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of E. coli from extra-intestinal sources were tested for susceptibility to non-beta-lactam drugs, and their serotypes were determined. Restriction enzyme digestion, followed by PFGE of DNA purified from isolates, was used to classify the phylogenetic relationship between them. Plasmid DNA from five isolates of different serotypes was used to transform an E. coli laboratory strain. The plasmids were partially sequenced. Results E. coli isolates from 86 patients, mostly elderly, were shown to be positive for inhibitor-susceptible ESBLs, and more resistant to cefotaxime than to ceftazidime. Ninety-six per cent of ESBL producers were also resistant to gentamicin, and 100% to trimethoprim/sulfamethoxazole and ciprofloxacin. All isolates belonged to one of five serotypes. PFGE analysis of purified DNA yielded 17 profiles. Sequencing of plasmids isolated from the transformants identified sul1, aac(6')-Ib and bla(CTX-M-2). These genes were embedded in an integron, InS21. Conclusions Extra-intestinal infections with ESBL-producing E. coli of different serotypes and probably mixed clonality showed a surprising homogeneity in resistance profiles, with 100% being co-resistant to ciprofloxacin and trimethoprim/sulfamethoxazole, and 96% to gentamicin. Plasmid DNA from three isolates from different serotypes contained integron InS21, previously demonstrated in Salmonella enterica from Argentina. This is the first molecular identification of an ESBL gene and integron in Israel or neighbouring geographical areas.

Polymorphisms in the rifampin resistance mutation frequency ( f ) were studied in 696 Escherichia coli strains from Spain, Sweden, and Denmark. Of the 696 strains, 23% were weakly hypermutable (4 × 10 −8 ≤ f < 4 × 10 −7 ), and 0.7% were strongly hypermutable ( f ≥ 4 × 10 −7 ). Weak mutators were apparently more frequent in southern Europe and in blood isolates (38%) than in urinary tract isolates (25%) and feces of healthy volunteers (11%).

The characteristics of Vibrio cholerae O1 biotype El Tor, serotype Ogawa isolates from outbreaks of cholera in 1998 amongst migrant workers in the South African provinces of Gauteng and Mpumalanga, on the border of Mozambique, are reported. The isolates seem to have originated from the same clone since they are of two closely related BglI ribotypes. These ribotypes had a high similarity to ribotypes of V. cholerae O1 recently found in three South-east Asian countries. Isolates were resistant to furazolidone, streptomycin, sulfamethoxazole, trimethoprim and tetracycline. Only two isolates contained plasmids of 54 and 63 kb in size. PCR and DNA sequencing revealed that the chromosomally located resistance determinants present included an aadA2 gene cassette contained in a class 1 integron; the SXT element, which is a transposon-like element containing resistance genes; and the tetA gene. A co-transfer of chromosomal closely located genes encoding the SXT element and tetA was shown by mating experiments, PCR and pulsed-field gel electrophoresis analyses. Our study shows for the first time that multiple-resistant V. cholerae O1 isolates containing class 1 integrons and the SXT element were responsible for cholera outbreaks in Southern Africa. Studies are needed to determine the spread of this multiple-resistant O1 strain and further genetic details of the association of the SXT element, tetA and class 1 integrons, including their means of transfer.

During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacter freundii in a Danish county, when a total of 24 resistant C. freundii isolates was detected. In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2. Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods. This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3′′)-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron. The source of the strain remains unresolved. Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.

In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB , aadA2 , blaP1 , dfrA1 , and dfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; β-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2 resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among different V. cholerae O serotypes of mainly clinical origin in Thailand.

In total, 120 Escherichia coli isolates positive for one of the gentamicin resistance (GEN R ) genes aac ( 3)-II, aac(3)-IV or ant(2″)-I were tested for gentamicin susceptibility by the agar dilution method. Isolates positive for aac(3)-IV or ant(2″)-I had an MIC distribution of 8–64 mg/L, whereas isolates positive for aac(3)-II had MICs of 32 to >512 mg/L, suggesting a relationship between the distribution of MICs and the specific GEN R mechanism. The MIC distribution, regardless of the GEN R mechanism, was 8 - >512 mg/L, which supports the clinical breakpoint of MIC >4 mg/L suggested by EUCAST and questions the breakpoint recommended by the CLSI (≥16 mg/L).

The presence and genetic content of integrons was investigated in eight Salmonella enteritica Typhimurium DT104 isolates from different pig herds in Denmark. Two different integrons were identified using PCR and sequencing. Each of the integrons carried a single resistance cassette in addition to the sull and qacE delta 1 genes characteristic of integrons. The first integron encoded the ant (3")-1a gene that specified resistance to spectinomycin and streptomycin. The second contained the pse-1 beta-lactamase gene. All the multiresistant strains contained both integrons. The presence of these two integrons did not account for the total phenotypic resistance of all the isolates and does not exclude the presence of other mobile DNA elements.

The occurrence of sulphonamide resistance was investigated in 998 Escherichia coli isolates, obtained from pig faeces collected at slaughter, Danish pork collected at retail outlets and from faeces from healthy persons in Denmark. In total 18% (n = 35), 20% (n = 38) and 26% (n = 161) of the E. coli isolates obtained from humans, pork and pigs, respectively, were resistant to sulphonamide. All sulphonamide resistant E. coli isolates were investigated for the presence of sul1, sul2, sul3 and intI1 genes by PCR. The sul1 gene was detected in 40% (n = 14), 29% (n = 11) and 55% (n = 88) of the sulphonamide resistant isolates from humans, pork and pigs, respectively. The sul2 gene was detected in 80% (n = 28), 76% (n = 29) and 50% (n = 81) of isolates from humans, pork and pigs, respectively. None of the human isolates were PCR-positive for sul3, whereas sul3 was present in 5% of the pork isolates and 11% of the pig isolates. Of the 113 sul1 positive isolates, 97 carried the integron-associated integrase gene intI1. All 20 sul3 positive isolates were positive for intI1, and in 12 of these isolates sul3 was the only sulphonamide resistance gene detected. The origin of sul1 and sul2 found in isolates from healthy humans is speculative, but their spread from pigs to humans via the food chain is possible.

Salmonella enterica serotype Typhimurium definitive phage type 104 was isolated several times from the same patient over a period of 2 years. The strain developed reduced sensitivity to fluoroquinolones, and a mutation in the gyrA gene that is associated with reduced sensitivity to quinolones was identified.

Plectasin is the first defensin-type antimicrobial peptide isolated from a fungus and has potent activity against gram-positive bacteria. By using an experimental meningitis model, the penetration of plectasin into the cerebrospinal fluid (CSF) of infected and uninfected rabbits and the bactericidal activities in CSF of the plectasin variant NZ2114 and ceftriaxone against a penicillin-resistant Streptococcus pneumoniae strain (NZ2114 and ceftriaxone MICs, 0.25 and 0.5 μg/ml, respectively) were studied. Pharmacokinetic analysis showed that there was a significantly higher level of CSF penetration of NZ2114 through inflamed than through noninflamed meninges (area under the concentration-time curve for CSF/area under the concentration-time curve for serum, 33% and 1.1%, respectively; P = 0.03). The peak concentrations of NZ2114 in purulent CSF were observed ∼3 h after the infusion of an intravenous bolus of either 20 or 40 mg/kg of body weight and exceeded the MIC >10-fold for a 6-h study period. Treatment with NZ2114 (40 and 20 mg/kg at 0 and 5 h, respectively; n = 11) caused a significantly higher reduction in CSF bacterial concentrations than therapy with ceftriaxone (125 mg/kg at 0 h; n = 7) at 3 h (median changes, 3.7 log 10 CFU/ml [interquartile range, 2.5 to 4.6 log 10 CFU/ml] and 2.1 log 10 CFU/ml [interquartile range, 1.7 to 2.6 log 10 CFU/ml], respectively; P = 0.001), 5 h (median changes, 5.2 log 10 CFU/ml [interquartile range, 3.6 to 6.1 log 10 CFU/ml] and 3.1 log 10 CFU/ml [interquartile range, 2.6 to 3.7 log 10 CFU/ml], respectively; P = 0.01), and 10 h (median changes, 5.6 log 10 CFU/ml [interquartile range, 5.2 to 5.9 log 10 CFU/ml] and 4.2 log 10 CFU/ml [interquartile range, 3.6 to 5.0 log 10 CFU/ml], respectively; P = 0.03) after the start of therapy as well compared to the CSF bacterial concentrations in untreated rabbits with meningitis ( n = 7, P < 0.05). Also, significantly more rabbits had sterile CSF at 5 and 10 h when they were treated with NZ2114 than when they were treated with ceftriaxone (67% [six of nine rabbits] and 0% [zero of seven rabbits], respectively, at 5 h and 75% [six of eight rabbits] and 14% [one of seven rabbits], respectively, at 10 h; P < 0.05). Due to its excellent CSF penetration and potent bactericidal activity in CSF, the plectasin variant NZ2114 could be a promising new option for the treatment of CNS infections caused by gram-positive bacteria, including penicillin-resistant pneumococcal meningitis.

The main purpose of the study was to investigate the frequency of ESBL-producing E. coli and Klebsiella strains in the Greater Copenhagen area. Four collections of strains were investigated: A) 380 consecutive E. coli and Klebsiella isolates primarily from urine, B) 200 gentamicin-resistant E. coli and Klebsiella isolates primarily from urine, C) 210 consecutive E. coli isolates from blood cultures, and D) 68 cefuroxime-resistant E. coli and Klebsiella isolates primarily from urine. Only one strain per patient was included. Strains with a zone diameter for cefpodoxime

Sir, Salmonella enterica serovars Enteritidis and Typhimurium are the most frequent Salmonella serovars isolated from humans. However, a significant proportion of human Salmonella isolates belong to other serovars, for which antimicrobial susceptibility patterns are rarely reported. In Denmark, the frequency of isolation of these less frequent Salmonella serovars from human clinical samples has increased in recent years. They represented 30% of all human Salmonella isolates, which corresponded to an incidence of 10.5 per 100 000 inhabitants in 2005. From 2003 to 2005, 530 isolates belonging to these Salmonella serovars were tested for their antimicrobial susceptibility by broth microdilution (Sensititre; Trek Diagnostic Systems Ltd, East Grinstead, UK) at the Statens Serum Institut. These included 106 Salmonella Virchow, 95 Salmonella Newport, 91 Salmonella Stanley, 80 Salmonella Dublin, 53 Salmonella Hadar, 38 Salmonella Saintpaul, 32 Salmonella Derby, 20 Salmonella Uganda and 15 Salmonella Anatum obtained from sporadic clinical infections, isolated at the 16 clinical microbiology laboratories in Denmark. The MICs for these 530 human clinical Salmonella isolates were determined for ampicillin (1–32 mg/L), cefalotin (4–32 mg/L), chloramphenicol (2–64 mg/L), ciprofloxacin (0.03–4 mg/L), gentamicin (1–32 mg/L), nalidixic acid (8–64 mg/L), sulfamethoxazole (64–1024 mg/L), streptomycin (4–64 mg/L), tetracycline (2–32 mg/L) and trimethoprim (4–32 mg/L). The CLSI (formerly NCCLS) breakpoints were used. For ciprofloxacin, a breakpoint of 0.125 mg/L was also used, and for streptomycin, a breakpoint of 32 mg/L was used. Escherichia coli ATCC 25922 was used for quality control. The number of tested isolates and the percentages of resistance are reported in Table 1. Isolates belonging to some serovars, e.g. Salmonella Dublin, Salmonella Saintpaul and Salmonella Derby, were susceptible to most antimicrobials tested, whereas isolates belonging to other serovars showed much higher frequencies of resistance, e.g. Salmonella Virchow, Salmonella Newport and Salmonella Stanley. A high prevalence of multidrug resistance (resistance to four or more antimicrobial agents) was observed among isolates belonging to the following serovars: Salmonella Hadar (30%), Salmonella Virchow (25%), Salmonella Stanley (22%) and Salmonella Newport (19%). Resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline was present in Salmonella Newport (15%), Salmonella Stanley (8%) and Salmonella Virchow (1%). Resistance to these five antimicrobials could indicate the presence of the multidrug-resistant region Salmonella genomic island 1 (SGI1). This region is well described and common in Salmonella Typhimurium phage type DT104. It has also been found in other Salmonella serovars, e.g. Salmonella Newport. Of the tested Salmonella Hadar and Salmonella Virchow isolates, 87% and 80%, respectively, were resistant to nalidixic acid. However, these isolates were not resistant to ciprofloxacin when the CLSI breakpoint (4 mg/L) was used. When using the EUCAST breakpoint for ciprofloxacin (0.125 mg/L), a good consistency was observed between nalidixic acid and ciprofloxacin resistance frequencies, even though minor differences were

The primary infecting Escherichia coli strains from 156 women with community-acquired uncomplicated urinary tract infection (UTI) randomized to pivmecillinam or placebo and the E. coli strains causing UTI at two follow-up visits were typed using pulsed-field gel electrophoresis (PFGE). In the pivmecillinam treatment group PFGE showed that among patients having a negative urine culture at the first follow-up 77% (46/60) had a relapse with the primary infecting E. coli strain and 23% (14/60) had reinfection with a new E. coli strain at the second follow-up. Among patients having E. coli at the first follow-up PFGE showed that 80% (32/40) had persistence with the primary infecting E. coli strain, 15% (6/40) had reinfection with a new E. coli strain, and 5% (2/40) had different E. coli strains at the two follow-up visits (one had reinfection followed by relapse, and the other had persistence followed by reinfection). In the placebo group the majority had E. coli at the first follow-up. PFGE showed that among these patients 96% (50/52) had persistence with the primary infecting E. coli strain and 4% (2/50) had different E. coli strains at the two follow-up visits (both had persistence followed by reinfection). The finding that the majority of UTIs at follow-up are caused by the primary infecting E. coli strain supports the theory of a vaginal and rectal reservoir but could also support the recent discovery that E. coli strains are able to persist in the bladder epithelium despite appropriate antibiotic treatment, constituting a reservoir for recurrent UTI.

Resistance to fluoroquinolones in urinary tract infection (UTIs) caused by Escherichia coli is associated with multiple mutations, typically those that alter DNA gyrase and DNA topoisomerase IV and those that regulate AcrAB-TolC-mediated efflux. We asked whether a fitness cost is associated with the accumulation of these multiple mutations. Mutants of the susceptible E. coli UTI isolate Nu14 were selected through three to five successive steps with norfloxacin. Each selection was performed with the MIC of the selected strain. After each selection the MIC was measured; and the regions of gyrA , gyrB , parC , and parE , previously associated with resistance mutations, and all of marOR and acrR were sequenced. The first selection step yielded mutations in gyrA , gyrB , and marOR . Subsequent selection steps yielded mutations in gyrA , parE , and marOR but not in gyrB , parC , or acrR . Resistance-associated mutations were identified in almost all isolates after selection steps 1 and 2 but in less than 50% of isolates after subsequent selection steps. Selected strains were competed in vitro, in urine, and in a mouse UTI infection model against the starting strain, Nu14. First-step mutations were not associated with significant fitness costs. However, the accumulation of three or more resistance-associated mutations was usually associated with a large reduction in biological fitness, both in vitro and in vivo. Interestingly, in some lineages a partial restoration of fitness was associated with the accumulation of additional mutations in late selection steps. We suggest that the relative biological costs of multiple mutations may influence the evolution of E. coli strains that develop resistance to fluoroquinolones.

Molecular typing is an important tool in surveillance and outbreak investigations of human Salmonella infections. In this study, three molecular typing methods were used to investigate the discriminatory ability, reproducibility and the genetic relationship between 110 Salmonella enterica subspecies enterica isolates. A total of 25 serotypes were investigated that had been isolated from humans or veterinary sources in Denmark between 1995 and 2001. All isolates were genotyped by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). When making genetic trees, all three methods resulted in similar clustering that often corresponded with serotype, although some serotypes displayed more diversity than others. Of the three techniques, MLST was the easiest to interpret and compare between laboratories. Unfortunately the seven housekeeping genes used in this MLST scheme lacked diversity and the ability to discriminate between isolates were higher with both PFGE and AFLP. The discriminatory power of AFLP and PFGE were similar but PFGE fingerprints were both easier to reproduce, interpret and less time-consuming to analyze when compared to AFLP. PFGE is the therefore the preferred molecular typing method for surveillance and outbreak investigations, whereas AFLP is most useful for local outbreak investigations.

The ability of extended-spectrum beta-lactamase (ESBL)-producing and non-ESBL-producing Klebsiella pneumoniae strains to induce a respiratory burst in polymorphonuclear leukocytes (PMNLs) was investigated. Ninety ESBL-producing and 178 non-ESBL-producing Klebsiella pneumoniae isolates were serotyped and their ability to induce a respiratory burst in PMNLs tested by monitoring the cells' chemiluminescence (CL) response. The percentage of isolates inducing high levels of CL response (CL75%) was significantly higher among non-ESBL producers (52%) than among ESBL producers (32.2%) ( P0.0001; OR=3.396; 95%CI=2.036-5.664). The median CL response was significantly higher among the non-ESBL producers (76.9%) than among the ESBL producers (52.6%) ( P=0.034). The two groups did not differ in their ability to resist intracellular killing by PMNLs ( P0.05), with strains inducing high levels of CL response having significantly lower survival rates (31.8% vs. 42.4%) than strains inducing low levels of CL response (164% vs. 200%) ( P0.01). The frequencies of the K2 and the K25 serotypes were significantly higher among ESBL-producing strains (17.8% and 22.2%, respectively) than among the non-ESBL producers (6.2% and 1.7%, respectively) ( P=0.0057 and P0.0001). Of the 77 Klebsiella K serotypes, 71 were detectable among the non-ESBL producers, but only 24 were detectable among the ESBL producers. ESBL-producing Klebsiella pneumoniae strains might have a greater pathogenic potential by virtue of their ability to escape the phagocytic activity of PMNLs.

The objective of the study was to investigate antimicrobial resistance, plasmids and class 1 integrons in 150 Shigella strains isolated from patients with diarrhea in Vietnam. Most isolates were resistant to the majority of antimicrobial agents used for treatment in the isolation areas and 90% were resistant to three or more antibiotics. A total of 20 strains yielded class 1 integrons, which harbored oxa1, dfrA, orfF, and aadA gene cassettes. The most common gene cassette, aadA2, was always located closest to the 3' conserved segment of the integrons and oxa1 and dfrA closest to the 5' end. Plasmid profiles of the 20 class 1 integron-positive strains all contained more than one plasmid, and 14 different profiles were found. No correlation was found between species, antibiograms, plasmid profiles, or presence of class 1 integrons. Conjugation resulted in 25 transconjugants, which all were resistant to four or more antimicrobial agents and all harbored at least one plasmid (60 kb). Class 1 integrons were detected in 64% of the transconjugants. Phenotypic resistance pattern and plasmid profiles of the transconjugants seemed independent of the presence of an integron. Class 1 integrons seemed of less importance in phenotypic antibiograms and in transfer of resistance genes than conjugative plasmids.

Salmonella enterica Typhimurium DT104, 961368, a veterinary field isolate that encodes a chromosomal cluster of resistance genes as well as two integrons, was used to study the mobility of resistance cassettes (aadA2 and pse-1) and nonintegron-associated resistance determinants (chloramphenicol and tetracycline). A range of natural plasmids was used as targets for the translocation of resistance. Plasmids that acquired resistance from the DT104 chromosome were segregated by conjugation into Escherichia coli K12. Plasmids R751, R388, and RP4::Tn7 acquired several combinations of resistance determinant (including single cassettes) at frequencies comparable with transposition. RP4 and pOG660 did not acquire any determinants from DT104. Phenotypic and PCR-based analysis of all the transconjugants that were translocated-both cassettes and more complex combinations of determinants-was carried out to determinate the genetic content. Translocation to R751 and R388 was associated with the loss of the indigenous trimethoprim cassette to both plasmids and also acquisition of sulfonamide resistance by R751 and RP4::Tn7, which indicated movement of the 3' terminus of one or both of the DT104 integrons. Sequencing of the R751 transconjugants confirmed these findings and showed that the translocation of streptomycin and ampicillin cassettes was associated with the precise excision of dhfrIIc and orfD cassettes. Furthermore, the translocation of multiple determinants occurred by at least two mechanisms, one of which was likely to involve a circular intermediate analogous to a composite cassette. Instability was detected in some of the transconjugants. The implication of the findings for the dissemination of resistance among clinical isolates is discussed.

A total of 78 gentamicin-resistant Escherichia coli strains from swine (27) and cattle (51) were characterized by phenotypic resistance, presence of selected aminoglycoside resistance genes, class 1 integrons and gene cassettes, and pulsed-field gel electrophoresis. Gentamicin resistance was mainly encoded by the ant(2'')-I gene that was found in 76% of all the strains investigated, whereas the aac(3)-IIa gene was found in 14%. The ant(2'')-I gene was predominant in strains from cattle, whereas the porcine strains contained both ant(2'')-I, aac(3)-IIa, and the aac(3)-IVa genes. The pulsed-field gel electrophoresis (PFGE) investigation indicated a close clonal relationship in half of the bovine strains whereas the remaining E. coli were unrelated. Among the E. coli investigated, 20% contained class 1 integrons. Genes encoding resistance to trimethoprim (dhfrI, dhfrIb, and dhfrVII), gentamicin, tobramycin, and kanamycin (ant(2'')-Ia streptomycin and spectinomycin (ant(3'')-Ia) and streptothricin (sat1) were identified as gene cassettes. The most prevalent gene cassettes were ant(3'')-Ia (11 isolates) and the dhfrI (nine isolates).

The clonal relationship among Salmonella enterica serotype Typhimurium isolates from selected pig production units in Denmark was investigated by the pulsed field gel electrophoresis (PFGE) typing method to determine environmental survival and spread of Salmonella in different herds. Thirty-four Typhimurium isolated during 1996–1998 from porcine faeces and environmental samples from three pig farms designated 1, 3 and 5 were characterised by PFGE using two restriction enzymes. Farm 5 supplied piglets to farm 1 and the herds were located close to each other. Results of PFGE analysis showed both intra- and inter-relationships, i.e. identical PFGE patterns among the faecal and environmental isolates from farm 1 and farm 5. All the isolates from farm 3 irrespective of the source showed identical PFGE patterns, but were different from samples from farms 1 and 5. This study indicates spread between farms and survival of a farm-specific clone. Furthermore, identical PFGE patterns of isolates from piglet supplier and finisher herds indicate that the farrow-to-grower herd of farm 5 was sub-clinically infected prior to delivery to farm 1 and thereby caused the transmission of Salmonella.

The aadA genes, encoding resistance to streptomycin and spectinomycin, have been found as gene cassettes in different gram-negative and gram-positive bacterial species. The present study has revealed the sequence of a new gene, aadA5 , integrated as a gene cassette together with the trimethoprim resistance gene dfr7 in a class 1 integron. The integron was located on a plasmid and was identified in a pathogenic porcine Escherichia coli isolate.

A total of 136 isolates of Salmonella enterica serovar Typhimurium DT104 from Denmark ( n = 93), Germany ( n = 10), Italy ( n = 4), Spain ( n = 5), and the United Kingdom ( n = 9) were characterized by antimicrobial resistance analysis, plasmid profiling, pulsed-field gel electrophoresis (PFGE) with the restriction enzymes Xba I and Bln I, and analysis for the presence of integrons and antibiotic resistance genes. The isolates from Denmark were from nine pig herds, while the isolates from other countries were both of animal and of human origin. All but 10 isolates were resistant to ampicillin, chloramphenicol, spectinomycin, streptomycin, sulfonamides, and tetracycline. Five isolates from the United Kingdom and Spain were sensitive to all antibiotics examined, whereas four isolates from the United Kingdom and the United States were also resistant to one or more of the antibiotics, namely, gentamicin, neomycin, and trimethoprim. All but two strains had the same PFGE profiles when the Xba I restriction enzyme was used, while seven different profiles were observed when the Bln I restriction enzyme was used. Different dominating Bln I types were observed among European isolates compared with the types observed among those from the United States. All the isolates harbored common 95-kb plasmids either alone or in combination with smaller plasmids, and a total of 11 different plasmid profiles were observed. Furthermore, all but one of the multidrug-resistant isolates contained two integrons, ant (3")-Ia and pse-1 . Sensitive isolates contained no integrons, and isolates that were resistant to spectinomycin, streptomycin, and sulfonamides had only one integron containing ant (3")-Ia . When restriction enzyme Bln I was used, the 14 isolates from one of the nine herds in Denmark showed unique profiles, whereas isolates from the remaining herds were homogeneous. Among isolates from seven of nine herds, the same plasmid profile (95 kb) was observed, but isolates from two herds had different profiles. Thus, either PFGE (with Bln I) or plasmid profiling could distinguish isolates from three of nine pig herds in Denmark. The epidemiological markers (antimicrobial susceptibility testing, plasmid profiling, and PFGE) applied demonstrated high in vivo stability in the Danish herds. This may indicate that some different strains of multidrug-resistant S. enterica serovar Typhimurium DT104 have been introduced into Danish food animal herds. The presence of isolates from six different countries with similar profiles by PFGE with Xba I and highly homogeneous profiles by PFGE with Bln I indicate that multidrug-resistant S. enterica serovar Typhimurium DT104 has probably been spread clonally in these countries. However, some minor variation could be observed by using plasmid profiling and profiling by PFGE with Bln I. Thus, a more sensitive technique for subtyping of strains of DT104 and a broader investigation may help in elucidating the epidemiological spread of DT104 in different parts of the world.