Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics

In Escherichia coli K-12, amplifiable resistance to tetracycline, chloramphenicol, and other unrelated antibiotics was mediated by at least four spatially separated loci. Tetracycline-sensitive mutants were isolated by Tn5 insertional inactivation of an amplified multiply resistant strain. One of these, studied in detail, showed coordinate loss of expression of all other resistance phenotypes. The Tn5 element in this mutant mapped to 34 min on the E. coli K-12 linkage map. We have designated the locus marA (multiple antibiotic resistance). Tetracycline-sensitive mutants containing marA::Tn5 regained all resistance phenotypes at frequencies of 10(-8) to 10(-7) upon precise excision of Tn5. Moreover, a newly described tetracycline efflux system (A. M. George and S. B. Levy, J. Bacteriol. 155:531-540, 1983) was inactivated in tetracycline-sensitive mutants, but recovered in tetracycline-resistant revertants. In merodiploids, F-prime marA+ expressed partial or complete dominance over corresponding mutant chromosomal alleles. Dominance tests also established that a previously amplified host and a mutant marA allele were preconditions for the expression of phenotypic resistances.     

Expanding the bactericidal action of the food color additive phloxine B to gram-negative bacteria

Phloxine B (D&C red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 microg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.     

Multiple antibiotic resistance (mar) locus in Salmonella enterica serovar typhimurium DT104

In order to understand the role of the mar locus in Salmonella with regard to multiple antibiotic resistance, cyclohexane resistance, and outer membrane protein F (OmpF) regulation, a marA::gfp reporter mutant was constructed in an antibiotic-sensitive Salmonella enterica serovar Typhimurium DT104 background. Salicylate induced marA, whereas a number of antibiotics, disinfectants, and various growth conditions did not. Increased antibiotic resistance was observed upon salicylate induction, although this was shown to be by both mar-dependent and mar-independent pathways. Cyclohexane resistance, however, was induced by salicylate by a mar-dependent pathway. Complementation studies with a plasmid that constitutively expressed marA confirmed the involvement of mar in Salmonella with low-level antibiotic resistance and cyclohexane resistance, although the involvement of mar in down regulation of OmpF was unclear. However, marA overexpression did increase the expression of a ca. 50-kDa protein, but its identity remains to be elucidated. Passage of the marA::gfp reporter mutant with increasing levels of tetracycline, a method reported to select for mar mutants in Escherichia coli, led to both multiple-antibiotic and cyclohexane resistance. Collectively, these data indicate that low-level antibiotic resistance, cyclohexane resistance, and modulation of OMPs in Salmonella, as in E. coli, can occur in both a mar-dependent and mar-independent manner.     

Overexpression of marA, soxS, or acrAB produces resistance to triclosan in laboratory and clinical strains of Escherichia coli

Triclosan (Irgasan) is a broad spectrum antimicrobial agent used in handsoaps, toothpastes, fabrics, and plastics. It inhibits lipid biosynthesis in Escherichia coli , probably by action upon enoyl reductase (FabI) (McMurry L.M., Oethinger M. and Levy S.B. (1988) Nature 394, 531–532). We report here that overexpression of the multidrug efflux pump locus acrAB , or of marA or soxS , both encoding positive regulators of acrAB , decreased susceptibility to triclosan 2-fold. Deletion of the acrAB locus increased the susceptibility to triclosan approximately 10-fold. Four of five clinical E. coli strains which overexpressed marA or soxS also showed enhanced triclosan resistance. The acrAB locus was involved in the effects of triclosan upon both cell growth rate and cell lysis.     

marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli.

Stable chromosomal multiple-antibiotic-resistant (Mar) mutants of Escherichia coli, derived by exposing susceptible cells to low concentrations of tetracycline or chloramphenicol, express cross-resistance to structurally unrelated antibiotics. The entire resistance phenotype is reversed to susceptibility by insertion of transposon Tn5 into a locus, designated marA, near 34 min on the chromosome (A. M. George and S. B. Levy, J. Bacteriol. 155:541-548, 1983). Strains in which 39 kbp of chromosomal DNA, including marA, had been deleted were unable to produce Mar mutants. The deletion strain could be complemented in trans by introduction of intact marA+ on plasmid F'506. Junction fragments from a strain containing marA::Tn5 were cloned, exploiting kanamycin resistance on Tn5 for selection. They were used as probes to search a phasmid library of E. coli K-12 for recombinants containing the marA+ region. Two phasmids which contained regions hybridizing to this probe were identified and shown to complement delta marA in a deletion strain. From one phasmid, several marA-containing fragments were cloned: those of greater than or equal to 7.8 kbp restored the ability to form Mar mutants in a deletion strain. These Mar mutants were shown to be dependent on the cloned marA fragment. Chromosomal as well as recombinant Mar mutants showed increased expression of a marA-specific mRNA species of about 1.4 kb, which was barely or not detectable in wild-type strains. Exposure of mutants and, to a lesser extent, parental strains to tetracycline or chloramphenicol resulted in elevated levels of mRNA which hybridized to the marA probe. These results indicate that the marA locus is needed for production of Mar mutants and is regulated, responding to at least two antibiotics to which it controls resistance.     

Prevalence of the multiple antibiotic resistance operon (marRAB) in the genus Salmonella

The multiple antibiotic resistance operon (marRAB) is a member of the multidrug resistance (mdr) systems. Similar to other mdr systems, this operon when induced encodes resistance to structurally and functionally unrelated antibiotics. marRAB has been shown to be conserved in the family Enterobacteriaceae, but within the genus Salmonella certain species appeared to be lacking this operon. To investigate how conserved the marRAB operon was in Salmonella, 30 veterinary isolates were examined by PCR, Southern blot, and dot blot analysis. Using DNA primers based on the marRAB operon of S. typhimurium, a predicted 2.3-kb amplicon resulted after PCR in 16 of the 30 organisms. The 2.3-kb DNA band from S. enteritidis was cloned and sequenced and shown to possess 99% sequence homology to marRAB from S. typhimurium. Using a labeled marRAB gene probe from S. enteritidis, Southern blot and dot blot analysis confirmed the presence of the operon in all 30 Salmonella species examined. Furthermore, when these isolates were induced with low levels of either tetracycline or chloramphenicol, increased antimicrobial resistance was observed to structurally and functionally unrelated antibiotics. Thus, the widespread occurrence of the marRAB locus in this genus prescribes judicious use of antimicrobials to avoid induction of a mdr phenotype.     

Fluoroquinolone resistance in Salmonella serovars isolated from humans and food animals

Quinolone-resistant Salmonella enterica usually contain a mutation in gyrA within the region encoding the quinolone resistance determining region of the A subunit of DNA gyrase. These mutations confer substitutions analogous to Escherichia coli Ser83-->Phe and Asp87-->Gly or Tyr, or a novel mutation resulting in Ala119-->Glu or Val. Mutations in gyrB are rare, and no mutations in parC or parE have been described. Quinolone-resistant Salmonella can also be cross-resistant to other agents including chloramphenicol and tetracycline. Increased efflux has been demonstrated and for some strains this has been associated with increased expression of acrB. Mutation in soxR has also been shown for one isolate. Detection of low level resistance (minimum inhibitory concentrations <0.5 microg ml(-1)) to fluoroquinolones is proving an increasing problem in the treatment of invasive Salmonella infections.     

Antimicrobial resistance and class I integrons in Salmonella enterica isolates from wild boars and Bísaro pigs

The antibiotic resistance phenotype and genotype and the integron type were characterized in 58 Salmonella enterica isolates recovered from Bísaro pigs and wild boars (20 S. Typhimurium, 17 S. Rissen, 14 S. Enteritidis and 7 S. Havana). Most S. Typhimurium isolates (15/20 of Bísaro pigs and wild boars) showed ampicillin, chloramphenicol, streptomycin, tetracycline, sulfonamide, and amoxicillin-clavulanic acid resistances. Of the 17 S. Rissen isolates of both origins, 13 were resistant to ampicillin, tetracycline and trimethoprim-sulfamethoxazole. Among the S. Enteritidis isolates of Bísaro pigs, eight were nalidixic acid-resistant and three were sulfonamide-resistant. The tet(A) or tet(G) genes were detected in most tetracycline-resistant isolates. The intI1 gene was identified in 72.5% of S. enterica isolates in which the conserved region 3' of class 1 integrons (qacEΔ1+sul1) was also amplified, whereas none had the intI2 gene. The dfrA12+orfF+aadA2 gene cassette arrangement was found in the variable region of class 1 integrons in 14 S. Rissen isolates. Fifteen S. Typhimurium isolates had two integrons with variable regions of 1000 and 1200 bp that harbored the aadA2 and blaPSE-1 gene cassettes, respectively. In these isolates the floR and tet(G) genes were also amplified, indicative of the genomic island 1 (SGI1). Salmonella Typhimurium and S. Rissen of animal origin frequently show a multi-antimicrobial resistant phenotype, which may have implications in public health.     

Organic solvent tolerance and antibiotic resistance increased by overexpression of marA in Escherichia coli.

We previously reported that overexpression of the soxS or robA gene causes in several Escherichia coli strains the acquisition of higher organic solvent tolerance and also increased resistance to a number of antibiotics (H. Nakajima, K. Kobayashi, M. Kobayashi, H. Asako, and R. Aono, Appl. Environ. Microbiol. 61:2302-2307, 1995). Most E. coli strains cannot grow in the presence of cyclohexane. We isolated the marRAB genes from a Kohara lambda phage clone and cyclohexane-tolerant mutant strain OST3408. We found a substitution of serine for arginine at position 73 in the coding region of marR of OST3408 and designated the gene marR08. Our genetic analysis revealed that marR08 is responsible for the cyclohexane-tolerant phenotype. We observed that the marA gene on high-copy-number plasmids increased the organic solvent tolerance of E. coli strains. Furthermore, exposure of E. coli cells to salicylate, which activates the mar regulon genes, also raised organic solvent tolerance. Overexpression of the marA, soxS, or robA gene increased resistance to numerous antibiotics but not to hydrophilic aminoglycosides.     

表达SLTEC保护性抗原的重组猪霍乱沙门氏菌C500株的构建及生物学特性

摘要:【目的】 利用平衡致死系统构建表达产类志贺氏毒素大肠杆菌(Shiga-like toxin Escherichia coli , SLTEC)保护性抗原的减毒猪霍乱沙门氏菌。【方法】 构建表达SLT-IIeB－FedF的重组质粒 ,再将其电转入终宿主菌减毒猪霍乱沙门氏菌ΔasdC500株中构建成口服活疫苗株 ,经聚丙烯酰胺凝胶电泳检测SLT-IIeB－FedF融合蛋白的表达情况,并观察重组菌体外培养的稳定性。【结果】 　利用宿主-载体平衡致死系统构建了表达SLTEC保护性抗原的重组减毒猪霍乱沙门氏菌     

Penetration of Salmonella through a polarized Madin-Darby canine kidney epithelial cell monolayer

Many intracellular parasites are capable of penetrating host epithelial barriers. To study this process in more detail we examined the interactions between the pathogenic bacteria Salmonella choleraesuis and polarized epithelial monolayers of Madin-Darby canine kidney (MDCK) cells grown on membrane filters. Association of bacteria with the MDCK cell apical surface was an active event, requiring bacterial RNA and protein synthesis, and was blocked by low temperatures. Salmonella were internalized within a membrane-bound vacuole and exhibited penetration through, but not between MDCK cells. A maximum of 14 Salmonella per MDCK cell crossed the monolayer per hour to the basolateral surface yet the monolayer remained viable and impermeable to Escherichia coli. Apical S. choleraesuis infection resulted in an increase in paracellular permeability but the MDCK intercellular contacts were not significantly disrupted. Basolateral S. choleraesuis infection was inefficient, and only small numbers of S. choleraesuis penetrated to the apical medium.     

Genetic analysis of multiple antimicrobial resistance in Salmonella isolated from diseased broilers in Egypt

To date, no information has been available on the molecular bases of antimicrobial resistance in Salmonella spp. from poultry in Egypt or even in Africa. Therefore, the objective of this study was to analyze, at the molecular level, the mechanisms of multidrug-resistance in isolates of Salmonella recovered from diseased broilers in Egypt. Twenty-one Salmonella isolates were identified; 13 of these isolates were Salmonella enterica serovar Enteritidis and eight Salmonella enterica serovar Typhimurium. 17 (81%). Salmonella isolates displayed multidrug resistance phenotypes, particularly against ampicillin, streptomycin, spectinomycin, kanamycin, tetracycline, chloramphenicol, and trimethoprim/sulfamethoxazole. PCR and DNA sequencing identified class 1 integrons in nine (42.9%) isolates and class 2 integrons in three (14.3%) isolates. The identified resistance genes within class 1 integrons were aminoglycoside adenyltransferase type A, aadA1, aadA2 and aadA5 and dihydrofolate reductase type A, dfrA1, dfrA5, dfrA12, dfrA15 and dfrA17. The β-lactamase encoding genes bla(TEM-1) and bla(CMY-2) and florfenicol resistance gene floR were also identified. Furthermore, the tetracycline resistance gene tet(A) was identified in 14 (66.7%) Salmonella isolates. To the best of our knowledge, this is the first report of the molecular basis of antimicrobial resistance in Salmonella spp. isolated from poultry in Africa.     

Comparison of the Rates of Motility of Salmonella with Those of Other Enteric Bacteria

Comparison of the rates of motility through a semisolid medium of 16 common Salmonella sp., 14 Escherichia coli serotypes, 4 Arizona strains, 2 Escherichia freundii (Citrobacter) isolates, 2 Proteus sp., and 2 Pseudomonas sp. revealed the following. (i) Very closely related bacteria could demonstrate markedly different rates of progression. (ii) All of the salmonellae tested advanced faster than the Proteus and Pseudomonas test cultures but some Salmonella sp., notably S. choleraesuis and S. typhi, progressed relatively slowly compared to many other test cultures. (iii) The mean rate of motility for the fastest 14 Salmonella sp. (1.49 cm/hr) was not statistically greater than the mean value for the 14 E. coli serotypes (1.31 cm/hr) at the 1% level of significance. Selective motility procedures may not be a reliable means of isolating all Salmonella sp. from materials contaminated with other bacteria.     

Induction of chloramphenicol and tetracycline resistance in Flexibacter sp. strain FS-1.

The gliding bacterium Flexibacter sp. strain FS-1 exhibits inducible resistance to chloramphenicol (Cmr) and tetracycline (Tcr). Either chloramphenicol or tetracycline alone induced a Cmr Tcr phenotype. The resistance is apparently not plasmid encoded.     

Molecular epidemiology of Salmonella enterica serovar typhimurium isolates from cattle in hokkaido, Japan: evidence of clonal replacement and characterization of the disseminated clone

The molecular epidemiology of 545 Salmonella enterica serovar Typhimurium isolates collected between 1977 and 2009 from cattle in Hokkaido, Japan, was investigated using pulsed-field gel electrophoresis (PFGE). Nine main clusters were identified from 116 PFGE patterns. Cluster I comprised 248 isolates, 243 of which possessed a sequence specific to definitive phage type 104 (DT104) or U302. The cluster I isolates were dominant in 1993 to 2003, but their numbers declined beginning in 2004. Beginning in 2002, an increase was observed in the number of cluster VII isolates, consisting of 21 PFGE patterns comprising 165 isolates. A total of 116 isolates representative of the 116 PFGE profiles were analyzed by multilocus variable-number tandem-repeat analysis (MLVA). Other than two drug-sensitive isolates, 19 isolates within cluster VII were classified in the same cluster by MLVA. Among the cluster VII isolates, an antibiotic resistance type showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, kanamycin, cefazolin, and sulfamethoxazole-trimethoprim and a resistance type showing resistance to ampicillin, streptomycin, sulfonamides, tetracycline, and kanamycin were found in 23 and 125 isolates, respectively. In the 19 isolates representative of cluster VII, the bla(TEM-1) gene was found on a Salmonella serotype Typhimurium virulence plasmid, which was transferred to Escherichia coli by electroporation along with resistance to two to four other antimicrobials. Genomic analysis by subtractive hybridization and plasmid analysis suggested that the bla(TEM-1)-carrying virulence plasmid has a mosaic structure composed of elements of different origin. These results indicate an emerging multidrug-resistant S. Typhimurium clone carrying a virulence-resistance plasmid among cattle in Hokkaido, Japan.     

The prevalence of Salmonella spp. in bovine faecal,rumen and carcass samples at a commercial abattoir

AIMS: To determine the prevalence, serotype and antibiotic resistance profile of Salmonella isolates in cattle and on carcasses at a commercial Irish abattoir. METHODS AND RESULTS: Faecal, rumen and carcass samples were collected from a beef abattoir over a 12-month period and examined for the presence of Salmonella spp. Isolates were serotyped, phage typed (when serotype was found to be S. Typhimurium) and tested for susceptibility to a panel of antibiotics. Salmonella was isolated from 2% of faecal, 2% of rumen and 7.6% of carcass samples. Salmonella was most frequently isolated from samples taken during the period August to October. S. Dublin was isolated from 72% of positive samples. S. Agona and S. Typhimurium definitive type (DT)104 were each isolated from 14% of positive samples. All S. Typhimurium DT104 isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulphafurazole and tetracycline (ACSSuT). On occasion, from a single animal, the same serotype was isolated from more than one sample (i.e. faeces and rumen; faeces and carcass; rumen and carcass; faeces, rumen and carcass). CONCLUSIONS: Salmonella is present in cattle at slaughter and on beef carcasses at an Irish abattoir, with a higher frequency of occurrence during the period August to October. Most isolates from the study are not commonly associated with human clinical infection, with the exception of S. Typhimurium DT104 (R-type ACSSuT). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides epidemiological data that is necessary for the understanding of beef as a source of human Salmonella infection.     

Retrospective and longitudinal study of salmonellosis in captive wildlife in Trinidad

Morbidity and mortality of captive wildlife at the Emperor Valley Zoo, Trinidad from 1993 to 1996 were analysed to determine involvement of Salmonella spp. A 6 mo longitudinal study was conducted to determine the frequency of isolation of Salmonella spp. from apparently healthy, sick and dead wild mammals, birds, and reptiles. The antibiograms of Salmonella isolates were determined using the disc diffusion method. Fecal samples randomly selected from animal enclosures and cloacal swabs of snakes were cultured for Salmonella spp. following enrichment in tetrathionate and selenite cystine broths. For the 1993-96 period, Salmonella spp. was implicated in 17 (12%) of 141 sick or dead animals and the predominant serotype was S. typhimurium. During the 6 mo prospective study in a mean animal population of 1,186, there were 20 (2%) and 14 (1%) animals that were sick and died respectively; Salmonella spp. was implicated in only one mortality. Overall, of 1,012 samples from apparently healthy wildlife cultured, 66 (7%) yielded 24 serotypes of Salmonella. The predominant serotype were S. seigburg (16 isolates), S. gaminara (6 isolates), and S. thompson (6 isolates). None of the samples yielded S. typhimurium. The frequency of isolation of Salmonella spp. in reptiles (14%) was significantly higher than found in either mammals (7%) or birds (3%). Sixty-five (99%) of 66 Salmonella spp. isolates exhibited resistance to one or more of the nine antimicrobial agents tested. Resistance was high to cephalothin (92%), moderate to streptomycin (35%) and tetracycline (29%), but significantly low to gentamicin (2%), chloramphenicol (0%), and sulphamethoxazole/trimethoprim (0%). The prevalence of asymptomatic infections by Salmonella spp. in zoo animals was high and the very high prevalence of antimicrobial resistance could be a problem when treating salmonellosis.     

High prevalence of extended-spectrum beta lactamases among Salmonella enterica Typhimurium isolates from pediatric patients with diarrhea in China

We investigated the extended-spectrum beta lactamases among 62 Salmonella enterica Typhimurium isolates recovered from children with diarrhea in a Chinese pediatric hospital. A large proportion of S. enterica Typhimurium isolates were resistant to multiple antimicrobial agents, including ampicillin (90.3%), tetracycline (80.6%), trimethoprim/sulfamethoxazole (74.2%), chloramphenicol (66.1%), cefotaxime (27.4%). Forty-nine (79.0%) of S. enterica Typhimurium isolates were positive for bla(TEM-1b) and resistant to ampicillin. Thirteen S. enterica Typhimurium isolates (21.0%) were positive for bla(CTX-M-1-group) and bla(CTX-M-9-group), and all isolates harboring bla(CTX-M) genes were positive for ISEcp1. Two main clones (PFGE type A and D) accounted for nearly 70% of S. enterica Typhimurium isolates, and 7 CTX-M-producing isolates belonged to PFGE type D. Collectively, our data reveal multi-drug resistance and a high prevalence of extended spectrum beta lactamases among S. enterica Typhimurium isolates from children in China. In addition, we report the first identification of bla(CTX-M-55) within Salmonella spp. Our data also suggest that clonal spread is responsible for the dissemination of S. enterica Typhimurium isolates.