Eradication of Salmonella and Arizona species from turtle hatchlings produced from eggs treated on commercial turtle farms.

On commercial turtle farms more than 40% of the hatchlings excrete detectable levels of Salmonella and Arizona spp. when hatched from nonsanitized eggs incubated in sawdust or dirt-filled chambers. Over a 3-year period on 10 farms, more than 10(6) turtle eggs were treated in an attempt to hatch Salmonella-free turtles. Eggs were sanitized in disinfectant, treated by temperature- or pressure-differential dip methods in solutions containing 500 micrograms or more of gentamicin sulfate per ml, and hatched in sanitized plastic chambers free of bedding material. The Salmonella and Arizona spp. infection levels for turtles produced from treated eggs were 0 and 1.12% for years 1 and 2, respectively, whereas infection levels for hatchlings produced from nontreated eggs during these periods were 47 and 44%, respectively. During year 3, dip solutions were filtered daily, treated at 100 degrees C for 15 min on a weekly basis to free the solution of microbial contaminants and egg protein, charged with gentamicin after 10,000 to 20,000 eggs had been treated to maintain antimicrobial activity at 500 micrograms/ml or more, and maintained at pH 6.0 to preserve optimal antimicrobial activity. The implementation of these measures in year 3 resulted in an infection level of 0.15% when the tissues of 3 of 1,959 hatchlings tested were positive for Salmonella and Arizona spp., whereas the tissues of 66 (49.0%) of 135 hatchlings produced from nontreated eggs were positive.     

Antibiotic marker modifications of lambda Red and FLP helper plasmids, pKD46 and pCP20, for inactivation of chromosomal genes using PCR products in multidrug-resistant strains

The Red recombinase system of bacteriophage Lambda has been used to inactivate chromosomal genes in bacteria using PCR products. In this study, we describe the replacement of the ampicillin resistance marker of helper plasmids pKD46 and pCP20 by a gentamicin resistance gene to disrupt chromosomal genes and then to eliminate FRT flanked resistance gene in multiple antibiotic-resistant Salmonella enterica strains.     

Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430

The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.     

Detection of apramycin resistant enterobacteriaceae in hospital isolates

Apramycin is a recently developed aminoglycoside restricted to veterinary therapy. Production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)-IV) conferring cross-resistance to this drug and to gentamicin was detected in 1984 in France in bacteria of bovine origin. This mechanism of resistance was apparently confined to animals. We have studied 17 strains resistant to apramycin and gentamicin isolated in 5 hospitals in Belgium. Conjugative plasmids encoding an AAC(3)IV were present in 14 isolates. Comparison of the restriction fingerprints revealed 6 different plasmid patterns: 8 plasmids belonged to 2 groups sharing extensive intragroup homology and 4 were not related to the other replicons. These results indicate dissemination of plasmids within and between hospitals, but also of the gene encoding an AAC(3)IV.     

Tobramycin,amikacin, sissomicin,and gentamicin resistant Gram-negative rods.

Sensitivities to gentamicin, sissomicin, tobramycin, and amikacin were compared in 196 gentamicin-resistant Gram-negative rods and in 212 similar organisms sensitive to gentamicin, mainly isolated from clinical specimens. Amikacin was the aminoglycoside most active against gentamicin-resistant organisms, Pseudomonas aeruginosa, klebsiella spp, Escherichia coli, Proteus spp, Providencia spp, and Citrobacter spp being particularly susceptible. Most of the gentamicin-resistant organisms were isolated from the urine of patients undergoing surgery. Gentamicin was the most active antibiotic against gentamicin-sensitive E coli, Proteus mirabilis, and Serratia spp. Pseudomonas aeruginosa and other Pseudomonas spp were most susceptible to tobramycin.     

Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates

Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000. Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only. Four carried tetY, and another four carried tetD. Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG. Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY. Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101. All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline. The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients. Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria. Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.     

大肠埃希菌氨基糖苷类耐药株 Aac(3) Ⅱ 基因保守区分析

常规方法分离鉴定47株大肠埃希菌,以标准纸片扩散法(K B法)对其进行常用氨基糖苷类药物敏感性测定,耐药株经PCR检测aac(3) Ⅱ基因保守区,扩增产物进行DNA测序分析。初步探讨大肠埃希菌氨基糖苷类抗生素耐药株与aac(3) Ⅱ基因保守区之间的关系,结果显示本地区大肠埃希菌氨基糖苷类抗生素耐药株的aac(3) Ⅱ基因保守区具65位G、84位T和65位A、84位C两种基因型,且高度耐药菌株皆为65位G、84位T基因型。Abstract:According to standard K B method,bacteriostatic tests were performed to screen out aminoglycoside resistance bacteria from 47 strains of isolated E.coli.To analyze correlations between the degree of E.coli aminoglycoside resistance and aac(3) Ⅱgene conserved region,PCR amplified aac(3) Ⅱgene conserved regions and were analyzed by DNA sequencing.The results showed that there were two species of aac(3) Ⅱgene type including 65G and 84T or 65A and 84C in the samples.Strains with high activity of modifying enzyme to gentamicin all were 65G and 84T aac(3) Ⅱgene type.     

Structural relationships among chloramphenicol-resistance plasmids of Staphylococcus aureus

Abstract Twelve different chloramphenicol-resistance (Cm^r) plasmids detected in Staphylococcus aureus strains isolated between 1952 and 1981 were characterized by restriction endonuclease, DNA hybridization and heteroduplex analyses. These studies revealed three families of Cm^r plasmids which were distinguished by their chloramphenicol acetyltransferase sequences; the prototype plasmids of the families were pC221, pC223 and pC194. The cat and replication ( rep ) genes of the plasmid pC221 were highly conserved in other pC221 family members and were related to their homologs in the pC223 family plasmids; however, the cat and rep genes of the pC194 family plasmids were distinct.     

The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan

Abstract The prevalence of aac(2')-Ia , a gene coding for gentamicin 2'-JV-acetyltransferase in Providenda stuartii , among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O -acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providenda rettgeri . Other bacteria that O -acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava . These data suggest that AAC(2')-Ia may contribute to the rO -acetylation of peptidoglycan in P. stuartii , but a more specific enzyme must also be produced for this function.     

Treatment of Salmonella-Arizona-Infected Turtle Eggs with Terramycin and Chloromycetin by the Temperature Differential Egg Dip Method

Attempts to eliminate Salmonella and Arizona infection from newly hatched turtles were made by dipping fresh eggs in cold solutions of Terramycin and Chloromycetin at 1,000, 1,200, 1,500 and 2,000 mug per ml for either 10, 20, or 30 min. Control groups consisted of hatchings produced from nondipped eggs or eggs dipped in chilled water. In two of the four experiments 5 to 10 eggs were blended on days 15, 30, and 45 post antibiotic dip treatment. Twenty-five to 60 hatchlings from each control or experimental dip groups were held in containers and the water was tested (excretion method) for Salmonella and Arizona every 15 or 30 days for 180 to 210 days after hatching. Representative turtles were homogenized (blending method) to determine if systemic infections were present. All specimens tested were enriched in tetrathionate and selenite cystine broth. Nondipped eggs and water-dipped eggs routinely showed Salmonella and Arizona present in egg homogenate and hatchlings emerging from these eggs excreted these pathogens. Terramycin- and Chloromycetin-dipped eggs were uniformly negative for these pathogens, only if fresh eggs were dipped. Bacteriological assay of container water and whole turtle homogenate from hatchlings were negative for Salmonella and Arizona if eggs were dipped in 1,000 mug of Terramycin early in the egg laying season or if eggs were dipped in 1,500 or 2,000 mug of Terramycin per ml late in the egg laying season. The results of temperature-differential egg dip studies suggest that this is a feasible and promising method by which to eradicate Salmonella and Arizona from the turtle.     

Occurrence of small Hsd plasmids in Salmonella typhi, Shigella boydii, and Escherichia coli.

The natural occurrence of small Hsd (host specificity for DNA) plasmids was demonstrated in restriction endonuclease-producing strains of Salmonella typhi, Shigella boydii, and Escherichia coli. The five Hsd plasmids isolated were between 5.0 and 12.2 kilobases long. The copy number of all the Hsd plasmids was high (more than 10 copies per cell). Introduction of these small plasmids into E. coli strain 0 drastically lowered the efficiency of plating of the lambda.0 phages (the efficiency of plating was less than 5 X 10(-5) PFU-1). High restriction endonuclease activities were detected in the Hsd plasmid-positive strains because of the elevated copy numbers of the hsdR+ gene. The advantages of using E. coli strains containing the small Hsd plasmids for purification of type II restriction endonucleases are discussed.     

Occurrence of Thermotolerant Campylobacter spp. on Eggshells: a Missing Link for Food-Borne Infections?

We analyzed the prevalence of thermotolerant Campylobacter spp. compared that of to Salmonella spp. in raw yolk and on eggshells. A total of 2,710 eggs were investigated for each bacterium. Viable bacteria were found in 4.1% (Campylobacter spp.) and 1.1% (Salmonella spp.) of the eggshell samples, whereas the egg yolk samples were negative for both bacteria.     

Salmonella spp. in the working environment of sewage treatment plants in Oslo, Norway.

The presence of Salmonella spp. was investigated at three sewage treatment plants in Oslo, Norway. Salmonella bacteria were isolated from floor surfaces and areas with hand contact in the treatment plant, from floor surfaces and areas with hand contact in the treatment plant, from floor surfaces in the changing rooms, and in one case from floor surfaces in an eating room. The sewage sludge contained from 140 to 140,000 Salmonella spp. per 100 g dry weight. Raw and treated sewage contained an average of 130 and 3 of these bacteria per 100 ml, respectively. There was poor correlation between the pattern of serotypes isolated from the sewage works and the patterns of those which were registered among the population of Oslo during the investigation. Neither enteropathogenic bacteria nor parasite eggs were found in fecal samples from employees at the plant. The health significance of the presence of Salmonella spp. in the environment of sewage workers is discussed.     

Genetic analysis of Escherichia coli urease genes: evidence for two distinct loci.

Studies with two uropathogenic urease-producing Escherichia coli strains, 1021 and 1440, indicated that the urease genes of each are distinct. Recombinant plasmids encoding urease activity from E. coli 1021 and 1440 differed in their restriction endonuclease cleavage sites and showed minimal DNA hybridization under stringent conditions. The polypeptides encoded by the DNA fragments containing the 1021 and 1440 urease loci differed in electrophoretic mobility under reducing conditions. Regulation of urease gene expression differed in the two ureolytic E. coli. The E. coli 1021 locus is probably chromosomally encoded and has DNA homology to Klebsiella, Citrobacter, Enterobacter, and Serratia species and to about one-half of the urease-producing E. coli tested. The E. coli 1440 locus is plasmid encoded; plasmids with DNA homology to the 1440 locus probe were found in urease-producing Salmonella spp., Providencia stuartii, and two E. coli isolates. In addition, the 1440 urease probe was homologous to Proteus mirabilis DNA.     

New tester strains of Salmonella typhimurium highly sensitive to mutagenic nitroarenes

The nitroreductase and acetyltransferase genes of Salmonella typhimurium TA1538 have been cloned into pBR322. When transformed into TA1538 derivatives, these plasmids provided the basis for an extremely sensitive assay for the detection of mutagenic nitroarenes.     

Amlodipine: a cardiovascular drug with powerful antimicrobial property

Ten cardiovascular drugs were procured in pure form from their manufacturers in India and screened for antimicrobial property against fifteen known bacteria belonging to both gram-positive and gram-negative types. These bacteria were inhibited by the common antibiotics at 1-5 mg ml(-1) level through our earlier studies. Since most of the bacteria were moderate to highly responsive to amlodipine, this compound was further tested in vitro against 504 bacteria comprising 4 genera of gram-positive and 15 genera of gram-negative bacteria. Most of these were inhibited by the drug at 50-200 microg ml(-1) level and few strains were sensitive even at lower concentrations (10 microg ml(-1)). The bacteria could be arranged in the decreasing order of sensitivity towards amlodipine in the following manner: Staphylococcus aureus, Vibrio cholerae, Vibrio parahemolyticus, Shigella spp., Salmonella spp., Bacillus spp., whereas Escherichia coli, Klebsiella spp. and Pseudomonas aeruginosa were found to be resistant to the lower concentrations of the drug. Amlodipine was found to be bactericidal in nature when its mode of action was studied against S. aureus 6571, V. cholerae 14035 and Sh boydii 8 NCTC 254/66. The antibacterial activity of amlodipine could also be confirmed in vivo. When it was given to Swiss strain of white mice at different dosages (30 and 60 microg/mouse), it could significantly protect the animals challenged with 50 MLD of Salmonella typhimurium NCTC 74. According to Chi square test the in vivo data were highly significant (p<0.001).     

Eradication of Arizona hinshawii from artificially infected turtle eggs.

Turtle eggs, 24 h old, were infected with Arizona hinshawii and treated 48 h later with gentamicin sulfate (Garasol; Shering Corp., Allantown, N.J.) by pressure differential egg dip treatment to ascertain the concentration of this reagent required to eradicate this pathogen from eggs. Infected eggs treated with 1,000 or 1,500 micrograms of gentamicin per ml of dip solution eliminated detectable A. hinshawii from eggs as determined by testing shells and embryo-yolk homogenates of 12-day-old eggs and the gastrointestinal tracts, kidneys, livers and gall bladders, and yolks of 50-day-old embryos. Treated eggs produced hatchlings which did not excrete detectable A. hinshawii at 72 h or 30 days after hatching, nor was this organism recovered from the visceral organs of these hatchlings when necropsied 30 days after hatching. Bacteriological assays on infected nontreated eggs showed that greater than 70% of the eggs harbored A. hinshawii, and eggs in this group produced hatchlings which actively excreted and harbored A. hinshawii. Eggs not infected or treated also produced turtles which excreted and systemically carried A. hinshawii and Salmonella spp. though not at the same level as did the turtles produced from infected, nontreated eggs.     

Eradication of Arizona hinshawii from artificially infected turtle eggs

Turtle eggs, 24 h old, were infected with Arizona hinshawii and treated 48 h later with gentamicin sulfate (Garasol; Shering Corp., Allantown, N.J.) by pressure differential egg dip treatment to ascertain the concentration of this reagent required to eradicate this pathogen from eggs. Infected eggs treated with 1,000 or 1,500 micrograms of gentamicin per ml of dip solution eliminated detectable A. hinshawii from eggs as determined by testing shells and embryo-yolk homogenates of 12-day-old eggs and the gastrointestinal tracts, kidneys, livers and gall bladders, and yolks of 50-day-old embryos. Treated eggs produced hatchlings which did not excrete detectable A. hinshawii at 72 h or 30 days after hatching, nor was this organism recovered from the visceral organs of these hatchlings when necropsied 30 days after hatching. Bacteriological assays on infected nontreated eggs showed that greater than 70% of the eggs harbored A. hinshawii, and eggs in this group produced hatchlings which actively excreted and harbored A. hinshawii. Eggs not infected or treated also produced turtles which excreted and systemically carried A. hinshawii and Salmonella spp. though not at the same level as did the turtles produced from infected, nontreated eggs.     

Vibrio mimicus diarrhea following ingestion of raw turtle eggs.

Clinical and epidemiological characteristics of diarrhea associated with Vibrio mimicus were identified in 33 hospitalized patients referred to the Costa Rican National Diagnostic Laboratory Network between 1991 and 1994. The relevant symptoms presented by patients included abundant watery diarrhea, vomiting, and severe dehydration that required intravenous Dhaka solution in 83% of patients but not fever. Seroconversion against V. mimicus was demonstrated in four patients, from whom acute- and convalescent-phase sera were obtained. Those sera did not show cross-reaction when tested against Vibrio cholerae O1 strain VC-12. All the V. mimicus isolates from these cases produced cholera toxin (CT) and were susceptible to commonly used antibiotics. Attempts to isolate this bacterium from stool samples of 127 healthy persons were not successful. Consumption of raw turtle eggs was recalled by 11 of the 19 (58%) individuals interviewed. All but two V. mimicus diarrheal cases were sporadic. These two had a history of a common source of turtle (Lepidochelys olivacea) eggs for consumption, and V. mimicus was isolated from eggs from the same source (a local market). Among the strains, variations in the antimicrobial susceptibility pattern were observed. None of the strains recovered from market turtle eggs nor the four isolates from river water showed CT production. Further efforts to demonstrate the presence of CT-producing V. mimicus strains in turtle eggs were made. Successful results were obtained when nest eggs were tested. In this case, it was possible to isolate CT- and non-CT-producing strains, even from the same egg. For CT detection we used PCR, enzyme-linked immunosorbent assay (ELISA), and Y-1 cell assay, obtaining a 100% correlation between ELISA and PCR results. Primers Col-1 and Col-2, originally described as specific for the V. cholerae O1 ctxA gene, also amplified a 302-bp segment with an identical restriction map from V. mimicus. These results have important implications for epidemiological surveillance in tropical countries where turtle eggs are used for human consumption, serving as potential sources of cholera-like diarrhea.