Antibiotic resistance and detection of β-lactamase in bacterial strains of Staphylococci and Escherichia coli isolated from foodstuffs

Seventy strains of Staphylococcus spp. and Escherichia coli (35 each) were isolated from various foodstuffs and identified on the basis of cultural, morphological and biochemical characteristics and were further tested for their antibiotic susceptibility with commonly used antibiotics/drugs. 69.2% of the strains of Staphylococci were resistant to co-trimazine and 34.6% were resistant to penicillin-G. 19.2% of the staphylococcal isolates exhibited resistance to cloxacillin, nalidixic acid, methicillin and tetracycline whereas 15.3% of the staphylococcal isolates were resistant to amoxycillin and nitrofurantoin. The isolated E. coli strains exhibited sharp peaks of resistance to antimicrobial agents such as tetracycline (72%), doxycycline (60%) and nalidixic acid (48%). Forty-four percent of the E. coli strains were resistant to nitrofurantoin and penicillin-G respectively. Among the 13 antibiotics/drugs tested for resistance, six different resistance patterns were observed in staphylococcal isolates and seven different resistance patterns were observed in the E. coli isolates from various foodstuffs. Bacterial strains exhibiting MIC values 100 g/ml for ampicillin and cloxacillin were screened for -lactamase activity and out of 10 staphylococcal isolates, seven were found to be positive for -lactamase, whereas out of 13 E. coli isolates tested for -lactamase production, only three were found to be positive.     

Incidence of antimicrobial resistance among Enterobacteriaceae isolated in Riyadh

The antimicrobial resistance of 1,018 isolates of Enterobacteriaceae isolated from fecal specimens of the urban population of Riyadh, Saudi Arabia, was studied. Resistance to 1 or more of 10 antimicrobial agents was encountered in 50.2% of the isolates. Of the isolates tested,Escherichia coli (0.8%) andKlebsiella species (1.6%) were found resistant to seven antimicrobial agents simultaneously: ampicillin, chloramphenicol, kanamycin, colistin, streptomycin, tetracycline, and carbenicillin. Resistance to nalidixic acid was encountered in only 0.68% of theE. coli isolates. No isolate was found to be resistant to gentamicin. Eighty-six of the resistant strains were tested for their ability to transfer their resistance. Forty percent were able to do so withE. coli K-12.     

Antimicrobial susceptibility, β-lactamase and enterotoxin production in <Emphasis Type="Italic">Bacillus cereus</Emphasis> isolates from clinical and food samples

The antimicrobial susceptibility of 30 clinical and 30 food Bacillus cereus isolates was determined. All isolates were susceptible to streptomycin, ciprofloxacin and gentamicin, 90 % of them to clindamycin and vancomycin, and 67 % to erythromycin. All isolates were resistant to amoxicillin with clavulanic acid, ampicillin, cefotaxime, ciprofloxacin, cloxacillin, cefotaxime with clavulanic acid and penicillin. The MIC values (determined by E-tests) were 48–256 mg/L for ampicillin, 0.19–1.5 mg/L for gentamicin, 0.125–1.0 mg/L for clindamycin, 0.047–4.0 mg/L for erythromycin and 1.5–16 mg/L for vancomycin. The MICs 4.6–18.75 g/L were observed for penicillin using the microdilution method. The presence of metallo-β-lactamases was detected by E-test for 100 % of strains. Nonhemolytic diarrheal enterotoxin (NHE) was produced by 98.3 % of strains, while 31.7 % of them produced hemolytic diarrheal enterotoxin (HBL). Clinical isolates produced 10 % more HBL than food isolates. The psychrotrophic strains isolated from food samples produced NHE at 6.5 °C in 73 % of cases.     

Drug resistance in Indian isolates of enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis>

Five different strains of enteropathogenic Escherichia coli (EPEC) and one non-pathogenic strain of E. coli were studied for the determination of resistance to various antibiotics by disc inhibition test. Our results demonstrated multiple drug resistance among the selected E. coli isolates with all of them including the non-pathogenic control strain showing high resistance towards ampicillin. Our results from conjugation test clearly showed the presence of most of the antibiotic markers on the transferred plasmids. Simultaneously, the plasmid profile of conjugants also indicated the presence of more than one plasmid along with the expected megaplasmid of ~90 Kb present in them. Thus our results amply demonstrate that these drug markers are associated with these mobile plasmids.     

Antimicrobial resistance in Staphylococcus spp., Escherichia coli and Enterococcus spp. in dogs given antibiotics for chronic dermatological disorders, compared with non-treated control dogs

   

The aim of this study was to evaluate antimicrobial resistance in canine staphylococci, Escherichia coli and enterococci, which were isolated from 22 dogs with pyoderma and a history of previous antibiotic treatment, compared to bacterial isolates from 56 non-treated control dogs. Two isolates of each bacterial species per dog were investigated, if detected. Staphylococcal isolates from dogs with pyoderma (35 isolates) were more resistant to sulphatrimethoprim than the isolates from controls (56 isolates) (57% vs. 25%, p < 0.004). Multiresistance in staphylococci was also more common in dogs with pyoderma (29% vs. 9%, p = 0.02). A similar trend among isolates of E. coli was detected (24 and 74 isolates from treated and control dogs, respectively), but the differences were not significant. Resistance for macrolide-lincosamides was approximately 20% among staphylococci in both groups. Resistance to ampicillin among enterococci was 4%–7%. The age of the dogs might have an impact on resistance: multiresistance among staphylococcal isolates from younger dogs (≤5 years) was more common than in older dogs (≥6 years) (24%, vs. 0%, 63 and 27 isolates, respectively, p = 0.02). Staphylococci in younger dogs were more resistant to tetracycline (48% vs. 11%, p < 0.001) and sulphatrimethoprim (48% vs. 15%, p < 0.01) than those in older dogs. In contrast, the isolates of E. coli from older dogs tended to be more resistant, although a significant difference was detected only in resistance to tetracycline (13% vs. 2% of 40 and 50 isolates respecthely, p = 0.04)). The results of this small study indicate that resistance in canine staphylococci in the capital area of Finland is comparable with many other countries in Europe. Resistance in indicator bacteria, E. coli and enterococci, was low.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

Patterns of Antimicrobial Resistance Observed in Escherichia coli Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

A Possible Role of R Plasmids in Bacterial Permeability for β-Lactam Antibiotics

Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC-resistance levels of these strains were markedly reduced. Reduction of CER-resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, β-lactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains, the recipient strains of S. marcescens 13031 (rif), Escherichia coli K-12 (rif), and E. coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeability to these antibiotics was significantly lower than that of the original strain. This fact suggests the possibility that the R plasmid from strain 13093 may be involved not only in production of β-lactamases, but also in regulation of bacterial permeability for cephalosporins.     

Genotype Analysis of Escherichia coli Strains Isolated from Children and Chickens Living in Close Contact

Escherichia coli isolates from rectal swabs from 62 chickens and stools from 42 children living in close contact with chickens on the same farms in Kiambu district, Kenya, were compared for their genetic relatedness. Antibiotic susceptibility profiles broadly categorized isolates from the children and from the chickens into two separate clusters: the majority (144; 85.5%) of the E. coli isolates from children were multidrug resistant, while the majority (216; 87.1%) of the E. coli isolates from chickens were either fully susceptible or resistant only to tetracycline. Sixty- and 100- to 110-MDA plasmids were found to encode the transferable resistance to co-trimoxazole and tetracycline. HindIII restriction endonuclease digestion of the 60- and 100- to 110-MDA plasmids produced four distinct patterns for isolates from children and three distinct patterns for isolates from chickens. XbaI digestion of genomic DNA followed by pulsed-field gel electrophoresis (PFGE) analysis produced 14 distinct clusters. There were six distinct PFGE clusters among the isolates from children, while among the isolates from chickens there were seven distinct clusters. Only one PFGE cluster contained isolates from both children and chickens, with the isolates displaying an approximately 60% coefficient of similarity. This study showed that although several different genotypes of E. coli were isolated from children and chickens from the same farms, the E. coli strains from these two sources were distinct.     

Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum

Summary Hybrid plasmids were constructed by combining in vitro the Escherichia coli plasmid pGA22, which carries the genes determining resistance to kanamycin, tetracycline, chloramphenicol and ampicillin, with the cryptic plasmids, pCG1 and pCG2, of Corynebacterium glutamicum. The hybrid plasmids were introduced into C. glutamicum and E. coli and replicated in both hosts. They expressed all the E. coli resistance phenotypes except ampicillin resistance in C. glutamicum. The levels of antibiotic inactivating enzymes encoded on these plasmids were about four to ten times lower in C. glutamicum than in E. coli. Despite the lack of expression of ampicillin resistance, -lactamase activity was detected in C. glutamicum carrying hybrid plasmids.     

Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability

Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi^–, and the 44.5 Md Kc^r plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, transfer frequencies, and in vitro stability, were studied. Plasmids generally showed a wide host range and high stability in the transconjugants tested. It could be concluded that these plasmids may be widely disseminated in the environment studied.     

Prevalence of Escherichia coli strains resistance to antibiotics in wound infections and raw milk

Antibiotic-resistant Escherichia coli strains including extended-spectrum β-lactamase (ESBL) isolates are globally widespread in medical, food, and environmental sources. Some of these strains are considered the most pathogenic bacteria in humans. The present work examined the predominance of antibiotic resistance in E. coli strains in wound infections comparing with E. coli strains isolated from a raw milk as a potential source of those strains. The wound infections included abdomen, anus, arm, back, buttock, chest, foot, hand, head, leg, lung, mouth, neck, penis, thigh, toe, and vagina infections. In total, 161 and 153 isolates identified as E. coli were obtained from wound infections and raw milk, respectively. A Vitek 2 system innovated by bioMérieux, France was applied to perform the identification and susceptibility tests. The E. coli isolates that have ability to produce ESBL were detected by an ESBL panel and NO45 card (bioMérieux). Over half of the E. coli were from abdomen, back, and buttock wound infections. More than 50%of the E. coli isolates obtained from wound infections were resistant to cefazolin, ampicillin, cefuroxime, ciprofloxacin, mezlocillin, moxifloxacin, piperacillin, and tetracycline; 70% of the isolates from wound infections and 0% of the isolates from raw milk were E. coli isolates produced ESBL. The data showed that the strains resistance to multi-antibiotic and produced ESBL are more widespread among wound infections than in raw milk.     

Antimicrobial Resistance of Escherichia coli O157 Isolated from Humans, Cattle, Swine, and Food

A total of 361 Escherichia coli O157 isolates, recovered from humans, cattle, swine, and food during the years 1985 to 2000, were examined to better understand the prevalence of antimicrobial resistance among these organisms. Based on broth microdilution results, 220 (61%) of the isolates were susceptible to all 13 antimicrobials tested. Ninety-nine (27%) of the isolates, however, were resistant to tetracycline, 93 (26%) were resistant to sulfamethoxazole, 61 (17%) were resistant to cephalothin, and 48 (13%) were resistant to ampicillin. Highest frequencies of resistance occurred among swine isolates (n = 70), where 52 (74%) were resistant to sulfamethoxazole, 50 (71%) were resistant to tetracycline, 38 (54%) were resistant to cephalothin, and 17 (24%) were resistant to ampicillin. Based on the presence of Shiga toxin genes as determined by PCR, 210 (58%) of the isolates were identified as Shiga toxin-producing E. coli (STEC). Among these, resistance was generally low, yet 21 (10%) were resistant to sulfamethoxazole and 19 (9%) were resistant to tetracycline. Based on latex agglutination, 189 (52%) of the isolates were identified as E. coli O157:H7, among which 19 (10%) were resistant to sulfamethoxazole and 16 (8%) were resistant to tetracycline. The data suggest that selection pressure imposed by the use of tetracycline derivatives, sulfa drugs, cephalosporins, and penicillins, whether therapeutically in human and veterinary medicine or as prophylaxis in the animal production environment, is a key driving force in the selection of antimicrobial resistance in STEC and non-STEC O157.     

Moderate Prevalence of Antimicrobial Resistance in Escherichia coli Isolates from Lettuce,Irrigation Water,and Soil

Fresh produce is known to carry nonpathogenic epiphytic microorganisms. During agricultural production and harvesting, leafy greens can become contaminated with antibiotic-resistant pathogens or commensals from animal and human sources. As lettuce does not undergo any inactivation or preservation treatment during processing, consumers may be exposed directly to all of the (resistant) bacteria present. In this study, we investigated whether lettuce or its production environment (irrigation water, soil) is able to act as a vector or reservoir of antimicrobial-resistant Escherichia coli. Over a 1-year period, eight lettuce farms were visited multiple times and 738 samples, including lettuce seedlings (leaves and soil), soil, irrigation water, and lettuce leaves were collected. From these samples, 473 isolates of Escherichia coli were obtained and tested for resistance to 14 antimicrobials. Fifty-four isolates (11.4%) were resistant to one or more antimicrobials. The highest resistance rate was observed for ampicillin (7%), followed by cephalothin, amoxicillin-clavulanic acid, tetracycline, trimethoprim, and streptomycin, with resistance rates between 4.4 and 3.6%. No resistance to amikacin, ciprofloxacin, gentamicin, or kanamycin was observed. One isolate was resistant to cefotaxime. Among the multiresistant isolates (n = 37), ampicillin and cephalothin showed the highest resistance rates, at 76 and 52%, respectively. E. coli isolates from lettuce showed higher resistance rates than E. coli isolates obtained from soil or irrigation water samples. When the presence of resistance in E. coli isolates from lettuce production sites and their resistance patterns were compared with the profiles of animal-derived E. coli strains, they were found to be the most comparable with what is found in the cattle reservoir. This may suggest that cattle are a potential reservoir of antimicrobial-resistant E. coli strains in plant primary production.     

Clonal Diversity of Escherichia Coli Isolates from Marketed Beef in East Malaysia

Summary Escherichia coli, including Shiga-like toxin producing E. coli (STEC), serogroup O157:H7 and E. coli O157, were isolated from raw beef marketed in Sarawak and Sabah, East Malaysia. Molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on 51 confirmed E. coli isolates. Of the 51 isolates, five were E. coli O157:H7, four E. coli O157, two non-O157 STEC and 40 other E. coli isolates (non-STEC). Digestion of chromosomal DNA from these E. coli isolates with restriction endonuclease XbaI (5′-TCTAGA-3′), followed by PFGE, produced 45 restriction endonuclease digestion profiles (REDPs) of 10–18 bands. E. coli O157:H7 isolates from one beef sample were found to have identical PFGE profiles. In contrast, E. coli serogroup O157 from different beef samples displayed considerable differences in their PFGE profiles. These suggested that E. coli isolates of both serogroups were not closely related. A large variety of PFGE patterns among non-STEC isolates were observed, demonstrating a high clonal diversity of E. coli in the beef marketed in East Malaysia. The distance matrix values (D), calculated showed that none of the pathogenic E. coli strains displayed close genetic relationship with the non-STEC strains. Based on the PFGE profiles, a dendrogram was generated and the isolates were grouped into five PFGE clusters (A–E). From the dendrogram, the most related isolates were E. coli O157:H7, grouped within cluster B. The STEC O157:H7 beef isolates were more closely related to the clinical E. coli O157:H7 isolate than the E. coli O157:H7 reference culture, EDL933. Cluster A, comprising many of other E. coli isolates was shown to be the most heterogeneous. PFGE was shown to possess high discriminatory power in typing pathogenic and non-pathogenic E. coli strains, and useful in studying possible clonal relationship among strains.     

Occurrence and transferability of β-lactam resistance inEnterobacteriaceae isolated inChildren's University Hospital in Bratislava

Occurrence and transferability of β-lactam resistance in 30 multi-resistantEscherichia coli, Klebsiella spp.,Enterobacter spp.,Pantoea agglomerans, Citrobacter freundii andSerratia marcescens strains isolated from children between 0 and 3 years of age is presented. The strains were resistant to ampicillin (30), cefoxitin (22), cefotaxime (30), ceftriaxone (30), ceftazidime (30) and aztreonam (28), but susceptible to cefepime (30) and imipenem (26). Twenty-eight of 30 isolates possessed a transferable resistance confirmed by conjugation and isolation of 79–89-kb plasmids. The β-lactam resistance was due to production of β-lactamases and ceftazidime proved to be stronger β-lactamase inductor than ceftriaxone. Twenty-five clinical isolates expressed transferable extended spectrum β-lactamases, and chromosomally encoded AmpC β-lactamase.     

Multidrug-Resistant Enterococci in Animal Meat and Faeces and Co-Transfer of Resistance from an <Emphasis Type="Italic">Enterococcus durans</Emphasis> to a Human <Emphasis Type="Italic">Enterococcus faecium</Emphasis>

Forty-eight isolates resistant to at least two antibiotics were selected from 53 antibiotic-resistant enterococci from chicken and pig meat and faeces and analysed for specific resistance determinants. Of the 48 multidrug-resistant (MDR) strains, 31 were resistant to two antibiotics (29 to erythromycin and tetracycline, 1 to erythromycin and vancomycin, 1 to vancomycin and tetracycline), 14 to three (erythromycin, tetracycline and vancomycin or ampicillin) and 3 to four (erythromycin, vancomycin, ampicillin and gentamicin). erm(B), tet(M), vanA and aac (6′)-Ie aph (2′′)-Ia were the antibiotic resistance genes most frequently detected. All 48 MDR enterococci were susceptible to linezolid and daptomycin. Enterococcus faecalis (16), Enterococcus faecium (8), Enterococcus mundtii (2) and Enterococcus gallinarum (1) were identified in meat, and E. faecium (13) and Enterococcus durans (13) in faeces. Clonal spread was not detected, suggesting a large role of gene transfer in the dissemination of antibiotic resistance. Conjugative transfer of resistance genes was more successful when donors were enterococcal strains isolated from faeces; co-transfer of vanA and erm(B) to a human E. faecium occurred from both E. faecium and E. durans pig faecal strains. These data show that multidrug resistance can be found in food and animal species other than E. faecium and E. faecalis, and that these species can efficiently transfer antibiotic resistance to human strains in inter-specific matings. In particular, the occurrence of MDR E. durans in the animal reservoir could have a role in the emergence of human enterococcal infections difficult to eradicate with antibiotics.     

Occurrence and transmission of class 1 and 2 integrons among phenotypic highly ampicillin-resistant avian <Emphasis Type="Italic">Escherichia coli</Emphasis> isolates from Pakistan

Thirty four avian Escherichia coli isolates were collected from different cities of Punjab province, Pakistan during 2008–2009. Twenty five phenotypic highly ampicillin-resistant (MICs ≥ 256 μg/ml) avian E. coli strains were selected for the investigation of occurrence and transmission of class 1, 2 and 3 integrons and β-lactamase genes. Amoxicillin, sulfonamide, trimethoprim, enrofloxacin, pefloxacin and tetracycline were the most common phenotypic resistant among ampicillin-resistant avian E. coli strains. Integrons and β-lactamase were found 60 and 72% respectively. Class 1 and 2 integrons were found 52 and 8%, while class 3 integrons were not found in all strains. All class 1 positive strains had variable fragments associated with gene cassettes dfrA7, dfrA1-aadA1, aadA1, aadA22 and dfrA12-orfF-aadA2 respectively, which confer resistance to trimethoprim and streptomycin. Class 2-positive strains had similar gene cassettes array dfrA1-sat1-aadA1 conferring resistance to trimethoprim, streptothricin and spectinomicin/streptomycin. Integrons are frequently found in β-lactamase positive isolates and widely disseminate multidrug resistance genes but they do not play role in the spreading of β-lactamase genes. Class 1 integrons gene cassette aadA22 is reported for the first time in avian E. coli. Findings of this study may provide important and useful information reflecting specific antibiotic selective pressure in Punjab province, Pakistan.     

Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater

Six copper-resistant bacterial strains were isolated from wastewater of tanneries of Kasur and Rohi Nala. Two strains tolerated copper at 380 mg/L, four up to 400 mg/L. Three strains were identified as members of the genusSalmonella; one strain was identified asStreptococcus pyrogenes, one asVagococcus fluvialis and the last was identified asEscherichia coli. The pH and temperature optimum for two of them were 7.0 and 30 °C, respectively; four strains had corresponding optima at 7.5 and 37 °C, respectively. All bacterial isola-tes showed resistance against Ag⁺ (280–350 mg/L), Co²⁺ (200–420), Cr^VI (280–400), Cd²⁺ (250–350), Hg²⁺ (110–200), Mn²⁺ (300–380), Pb²⁺ (300–400), Sn²⁺ (480–520) and Zn²⁺ (300–450). Largesized plasmids (>20 kb), were detected in all of the strains. After the isolates were cured of plasmids with ethidium bromide, the efficiency of curing was estimated in the range of 60–90%. Reference strain ofE. coli was transformed with the plasmids of the bacterial isolates which grew in Luria-Bertani medium containing 100 mg/L Cu²⁺. The capability to adsorb and afterwards accumulate Cu²⁺ inside their cells was assayed by atomic absorption spectrophotometer; all bacterial cells had the ability to adsorb 50–80 % of the Cu²⁺ and accumulate 30–45 % Cu²⁺ inside them after 1 d of incubation.     

Generation of Novel Plasmids in Escherichia coli S17-1(pSUP106)

When the highly metal-resistant acidophilic heterotrophic strain, Acidiphilium symbioticum KM2, was incubated with two Escherichia coli strains, viz. S17-1 (pSUP106) and K12, on a medium that supported growth of these two divergent species of different habitats, E. coli transconjugants were isolated that contained novel plasmids and were resistant to Zn²⁺ (48 mM), Cu²⁺ (12 mM), Ni²⁺ (12 mM), chloramphenicol (50 μg/ml), and tetracycline (25 μg/ml). The transconjugant plasmids did not hybridize with any of the A. symbioticum KM2 plasmids. After curing of the plasmids, the transconjugants became sensitive to 12 mM Zn²⁺, 12 mM Cu²⁺, and 12 mM Ni²⁺, but remained chloramphenicol and tetracycline resistant—the phenotypic markers that were originally present in pSUP106. That a part of pSUP106 was integrated into the chromosome of the transconjugants was evident from the hybridization of pSUP106 with chromosomal DNA of the cured derivatives of the transconjugants. Further, the transconjugant plasmids hybridized only with the chromosomal DNA of E. coli S17-1 and not with the chromosomal DNA of A. symbioticum KM2 or E. coli K12, suggesting their host chromosomal origin. Thus, the present study describes a unique event of genetic rearrangements in the E. coli strain S17-1 (pSUP106), resulting in the formation of novel plasmids conferring metal-resistance phenotypes in the cell. Received: 5 April 2002 / Accepted: 5 July 2002