A Conjugative Transfer System for the Rumen Bacterium, Butyrivibrio fibrisolvens, Based on Tn916-Mediated Transfer of the Staphylococcus aureus Plasmid pUB110

A limitation of genetic studies of the rumen bacterium, Butyrivibrio fibrisolvens, has been the availability of suitable vectors and transfer systems. Using the conjugative tetracycline resistant transposon, Tn916, the Staphylococcus aureus plasmid, pUB110, and the pUB110-based shuttle vector, pUBLRS, a conjugative transfer system was developed for B. fibrisolvens. B. fibrisolvens donor strains H17c2 and H17c12, containing Tn916 and pUB110 or pUBLRS, respectively, were used in mating experiments with selected B. fibrisolvens strains. Kanamycin resistant transconjugants, containing pUB110, of strains 193, 194, and 195 were detected at a combined average frequency of 7.78 × 10^-7 per donor and 1.11 × 10^-5 per recipient. Transconjugants of strains 193 and 194, containing pUBLRS, were detected at an average frequency of 1.22 × 10^-6 per donor and 4.70 × 10^-8 per recipient. Southern hybridization analysis confirmed the presence of pUB110 and pUBLRS in transconjugants. Results indicated that Tn916 was necessary for mobilization of pUB110 as transconjugants were not detected when the transposon was absent from the donor strains. The ability to mobilize pUB110 and pUBLRS between B. fibrisolvens strains provides a conjugative transfer system that circumvents problems encountered with electroporation.     

Response of RP1+ and RP1− strains ofEscherichia coli to antibacterial agents and transfer of resistance toPseudomonas aeruginosa

The sensitivity of strains ofEscherichia coli, with and without the RP1 R-factor, to antibiotics and other antibacterial agents has been studied. RP1⁺ strains ofE. coli were resistant to kanamycin, carbenicillin, and tetracycline, resistance to the first two antibiotics being produced by destruction of the drugs. This resistance could be transferred to two strains ofPseudomonas aeruginosa. The parent strain ofE. coli UB 1005, its two mutant strains (DC2 and DC3), and two of the strains with the RP1 R-factor showed a similar order of sensitivity to phenylmercuric nitrate, chlorhexidine, thiomersal, and mercuric chloride.E. coli strains DC2 and DC2 (RP1⁺) were the most sensitive to benzalkonium chloride and cetrimide. RP1⁺ strains were more resistant than RP1⁻ strains to lysozyme-ethylenediaminetetraacetic acid, but treatment of the former strains with acriflavine rendered the cells more sensitive to the lytic system. There was no evidence thatP. aeruginosa (RP1⁺) strains possessed increased resistance to polymyxin or to disinfectants, although they became somewhat less sensitive to lysozyme-ethylenediaminetetraacetic acid.     

Characterizing bacterial resistance to preservatives and disinfectants

Bacterial resistance to preservatives and disinfectants is a problem with serious economic and health consequences. Understanding the basis of resistance may lead to strategies or chemistries capable of reversing or subverting the mechanism. A collection of bacterial isolates resistant to dimethoxydimethyl hydantoin, glutaraldehyde, methylchloroisothiazolone/methylisothiazolone, and benzisothiazolone, as well as combination preservatives, has been gathered. In addition, a perusal of the literature also reveals reports of resistance to quaternary ammonium compounds, biguanides, iodophors, and peroxides, suggesting no particular chemistry is immune to resistance development. The majority of isolates in the collection are members of the genera Pseudomonas and Burkholderia, along with several representatives of the genus Alcaligenes and Enterobacter. Characterization of their susceptibility to other preservatives and disinfectants revealed several patterns of cross-resistance. All isolates were resistant to biocides other than the selecting compound. Several isolates were cross-resistant to either a quaternary ammonium compound or hydrogen peroxide; one P. fluorescens isolate was cross-resistant to both disinfectants. The presence of cross-resistance among preservatives and disinfectants has serious implications for the ability to eradicate resistant microbes from contaminated surfaces or manufacturing processes using commonly accepted disinfectants.     

Specificity of mutations induced by riboflavin mediated photosensitization in the supF gene of Escherichia coli

Riboflavin-mediated photosensitization has been shown to produce 8-hydroxyguanine (oh⁸Gua) in DNA. We investigated the specificity of mutation of photosensitized supF gene induced in Escherichia coli. The oh⁸Gua repair deficient E. coli mutant mutM and mutY were transformed with plasmid pUB3 carrying the supF gene irradiated with white light in the presence of riboflavin. Under these conditions, riboflavin photosensitization increased the amounts of oh⁸Gua in pUB3 DNA. Three types of a single base substitution occurring at G:C pairs were detected in both wild-type and mutM mutant strains. Almost all base substitutions were transversions to T:A or C:G pairs occurring at a similar extent in both wild-type and mutM strains. Mutations derived from mutY strain transformed with photosensitized DNA were only G:C to T:A transversions. These G:C to T:A transversions observed in the mutY strain were suggested to be the result of mispairing of oh⁸Gua with adenine. Riboflavin-mediated photosensitization may also produce lesions on DNA causing G:C to C:G changes by unknown mechanisms.     

Mercury Volatilization by R Factor Systems in <Emphasis Type="Italic">Escherichia coli</Emphasis> Isolated from Aquatic Environments of India

Ten Escherichia coli strains isolated from five different aquatic environments representing three distinct geographical regions of India showed significantly high levels of tolerance to the inorganic form of mercury, i.e., mercuric chloride (HgCl₂). MRD14 isolated from the Dal Lake (Kashmir) could tolerate the highest concentration of HgCl₂, i.e., 55 g/mL, and MRF1 from the flood water of the Yamuna River (Delhi) tolerated the lowest concentration, i.e., 25 g/mL. All ten strains revealed the presence of a plasmid of approximately 24 kb, and transformation of the isolated plasmids into the mercury-sensitive competent cells of E. coli DH5 rendered the transformants resistant to the same concentration of mercury as the wild-type strains. Mating experiments were performed to assess the self-transmissible nature of these promiscuous plasmids. The transfer of mercury resistance from these wild-type strains to the mercury-sensitive, naladixic acid-resistant E. coli K12 (F^–lac⁺) strain used as a recipient was observed in six of the nine strains tested. Transconjugants revealed the presence of a plasmid of approximately 24 kb. An evaluation of the mechanism of mercury resistance in the three most efficient strains (MRG12, MRD11, and MRD14) encountered in our study was determined by cold vapor atomic absorption spectroscopy (CV-AAS), and it was noted that resistance to HgCl₂ was conferred by conversion of the toxic ionic form of mercury (Hg⁺⁺) to the nontoxic elemental form (Hg⁰) in all three strains. MRD14 volatilized mercury most efficiently.     

Efficacy of some commerical disinfectants against the bacterial isolates from a poultry slaughterhouse in Turkey

In this study, efficacy of seven different commercial disinfectant preparations was investigated against characteristic bacteria of a poultry slaughterhouse in Ankara (Turkey) by using paper disc-agar diffusion method and surface effectiveness test. According to paper disc-agar diffusion method, some disinfectants had wide efficacy against the test bacteria. The disinfectant effectiveness generally increased by the increasing of disinfectant concentration. However, some disinfectants were ineffective even though at their highest concentrations. The most effective disinfectant was B which contains QAC as active agent.Staphylococcus spp.,Staphyloccus aureus andEscherichia coli were the most sensitive bacteria to the disinfectants. Some pathogenic isolates, especiallySalmonella spp. andCampylobacter spp., were the most resistant ones to many of the disinfectants tested. The results of paper disc-agar diffusion method indicated the importance of characteristic bacterial strains of food plants as the test bacteria for disinfectant efficacy tests. Conversely of paper disc-agar diffusion method, all disinfectants were effective against the isolates by surface effectiveness test depending on exposure time. The disinfectants, except A and F, produced at least 3 log unit reduction during 5 min of exposure. However, all disinfectants at their lowest concentrations were effective against all tested bacteria during 15 and 30 min by this test.     

Interaction of lead nitrate and cadmium chloride withEscherichia coli K-12 andSalmonella typhimurium global regulatory mutants

Summary To investigate the interactions of heavy metals with cells, a minimal medium for the growth of enteric bacteria using glycerol-2-phosphate as the sole phosphorus source was developed that avoided precipitation of Pb²⁺ with inorganic phosphate. Using this medium, spontaneous mutants ofEscherichia coli resistant to addition of Pb(NO₃)₂ were isolated. Thirty-five independent mutants all conferred a low level of resistance. Disk diffusion assays on solid medium were used to survey the response ofE. coli andSalmonella typhimurium mutants altered in global regulatory networks to Pb(NO₃)₂) and CdCl₂. Strains bearing mutations inoxyR andrpoH were the most hypersensitive to these compounds. Based upon the response of strains completely devoid of isozymes needed to inactivate reactive oxygen species, this hypersensitity to lead and cadmium is attributable to alteration in superoxide dismutase rather than catalase levels. Similar analysis of chaperonedefective mutants suggests that these metals damage proteins in vivo.     

Mercury Resistance in Bacterial Strains Isolated from Tailing Ponds in a Gold Mining Area Near El Callao (Bolívar State,Venezuela)

Bacterial resistance to mercury (Hg) was investigated in strains isolated from Hg-contaminated tailing ponds located in the gold mining area of El Callao (Bolívar State, Venezuela). High frequencies of resistance were detected to both inorganic-Hg and organomercurials among these strains. A broad range of resistance levels was observed when determining minimal inhibitory concentrations of Hg²⁺. Some strains were able to grow in liquid medium containing 25 μM Hg²⁺, whereas others grew at 300 μM Hg²⁺. Of 190 Hg-resistant strains tested, 58.2% were additionally shown to be resistant to ampicillin (40 mg/L), 33.3% to chloramphenicol (30 mg/L), 24.9% to streptomycin (30 mg/L), 23.3% to tetracycline (30 mg/L), and 1.6% to kanamycin (30 mg/L). Furthermore, we found that 20% of the Hg-resistant strains were simultaneously resistant to as many as four of these antibiotics, at the concentrations tested. The presence of large plasmids in 62.9% of 53 Hg-resistant strains screened prompted us to investigate the horizontal transfer of resistance determinants. Mating experiments were performed using Escherichia coli and Pseudomonas aeruginosa as recipient strains. The results obtained confirmed that indigenous Hg-resistant bacteria colonizing the tailing ponds can effectively transfer the phenotype to potentially pathogenic species.     

Transfer ofF′ fromEscherichia coli K 12 toEscherichia coli B and to strains ofParacolobacter andKlebsiella

The transfer of theF episome fromEscherichia coli K 12 toE. coli B,Paracolobacter andKlebsiella was studied. The frequency of transfer of the episomal markers toE. coli B was very low. The large majority ofE. coli B cells which had received the episomal markerslac ⁺ orgal ⁺ were F^–, which indicates that the episomal markers were stably integrated on the chromosome. Recombinants from K 12 F⁺ × B F^– crosses were mostly F^–. These results suggest that the multiplication of theF-factor ofE. coli K 12 is restricted inE. coli B. The transfer of theF-lac ⁺ Ad ⁺ episome fromE. coli K 12 toParacolobacter andKlebsiella strains was in most cases only possible when donor and acceptor strain were plated together on selective media. Stable incorporation of episomal markers was also found withParacolobacter coliforme. Paracolobacter aerogenoides andKlebsiella aerogenes strains could be infected withF-lac ⁺ Ad ⁺. The episomal markers were not incorporated and the episomes were easily lost, which indicates that these strains contained theF factor in the autonomous state.     

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.     

Tetracycline inhibition of changes in the redox potential during the logarithmic phase of growth of sensitive microorganisms

The addition of Oxytetracycline to an aerated broth culture of the sensitive strainsStaphylococcus Oxford andEscherichia coli 514 during the logarithmic phase of growth immediately stopped the usual steady drop in redox potential values. No differences were found in the dynamics of rH changes during growth of resistant variants of these strains.

     

Relationship Between the Persistence of mer Operon Sequences in Escherichia coli and Their Resistance to Mercury

Studies related to geographic distribution of E. coli carrying mer operon sequences were carried out on the Indian subcontinent. Out of the 80 E. coli isolates, collected from five geographically distinct regions of India, 68 were found to be resistant to one or the other heavy metal used in the study. Among these isolates, 36 were found to be resistant to the inorganic form (HgCl₂) and only 5 to resist both the inorganic and organic forms of mercury. Colony hybridization studies revealed 35 isolates out of 68 to hybridize with the probe. Interestingly, some of the mercury-sensitive isolates (Hg^s), especially from the Dal Lake, were found positive in hybridization studies. These findings, supported by mercury volatilization studies, indicate the presence of nonfunctional/vestigial mer sequences in the isolates collected from different environments. On the other hand, few of the mercury-resistant isolates (Hg^r) from the Yamuna River did not show any sign of hybridization. Further, volatilization studies also indicated an alternate mode of resistance mechanism operating in them. The studies demonstrate that the mer operon sequences share very high homology among the E. coli isolates collected from different geographical locations, and this metal resistance may be a genetic character that arose from a common ancestral background. Received: 25 May 2001 / Accepted: 27 June 2001     

Detection of<Emphasis Type="Italic">pap</Emphasis>-,<Emphasis Type="Italic">sfa</Emphasis>- and<Emphasis Type="Italic">afa</Emphasis>-specific DNA sequences in<Emphasis Type="Italic">Escherichia coli</Emphasis> strains isolated from extraintestinal material

P-fimbriae, S-fimbriae and AFA-adhesins are virulence factors responsible for adherence ofEscherichia coli strains to extraintestinal host-cell surface. Detection ofpap-,sfa- andafa-specific sequences performed by PCR revealed 74%pap ⁺, 65%sfa ⁺, and 8.3%afa ⁺ strains in a group of 84 extraintestialE. coli isolates. Detection in a group of fecal strains showed 29%pap ⁺, 21%sfa ⁺ and 4%afa ⁺ strains.pap together withsfa were found as the most frequent combination (56%) among extraintestinal isolates probably due to localization ofpap-andsfa-operons on a common pathogenicity island. The occurrence ofafa-specific sequence among 56 urine strains was 11%, although noafa ⁺ strain was detected among 28 gynecological isolates. No strains with detected adhesin operons were found among twenty (24%) extraintestinalE. coli strains.     

Efficacy of multiple metals against copper-resistant bacterial strains

Aims: The antibacterial efficacy of zeolites containing copper (Cu) or silver (Ag) ions or a combination was assessed against several reported copper‐resistant (Cu^R) bacterial strains. Methods and Results: Comparison strains were obtained from the American Type Culture Collection that had no documented metal resistance. Reductions in bacterial populations were determined after exposure time intervals of 3, 6 and 24 h. All three Cu^R strains of Salmonella enterica exhibited resistance to Cu, Ag and Cu/Ag after three and 6 h of exposure. Both the Cu^R and comparison strain of Enterococcus faecium were resistant to both metals and the metal combination. Cu^RPseudomonas putida was significantly reduced by all zeolites within 3 h. The Cu^REscherichia coli strain was more sensitive to Cu, but more resistant to Ag than the comparison strain; however, significant reductions were achieved within 3 h with both Cu and Cu/Ag, and within 24 h with Ag. Conclusions: Some strains with reported resistance to Cu were also resistant to Ag, suggestive of a shared resistance mechanism such as an indiscriminate Cu efflux pump. Ent. faecium appears to have innate resistance to both metals. In general, Ent. faecium was the most resistant species to the individual metals and the combination of metals, Ps. putida the least resistant, and the Salmonella strains were more resistant than E. coli. Significance and Impact of the Study: Several of the comparison strains with no reported copper resistance were resistant to one or both metals. This may call into question the methods for determining bacterial metal resistance, which typically use nutrient‐rich media containing metals to assess the ability of the bacteria to grow in comparison with a wild‐type strain. Nevertheless, all the Cu^R strains evaluated in this study, with the exception of Ent. faecium, were reduced using the Cu and Ag zeolite combination.     

Plasmid mediated metal and antibiotic resistance in marinePseudomonas

Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marinePseudomonas toEscherichia coli occurred during mixed culture incubation in liquid broth at 10^–4 to 10^–5 ml^–1. However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 intoE. coli competent cells was successful; however, the efficiency of transformation (1.49×10² Hg^r transformants g^–1 pMR1 DNA) was low.E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.     

Characterization of an extremely heat-resistant Escherichia coli obtained from a beef processing facility

Aims:  This study aimed to determine the survival of Escherichia coli strains during steam and lactic acid decontamination interventions currently used by the beef‐processing industry, and to determine their heat resistance. Methods and Results:  Strains were grouped into cocktails of five strains each differing in their RAPD patterns for subsequent identification. Steam and lactic acid treatments on meat reduced cell counts of E. coli strain cocktails by 90–99%. The 20 slaughter plant isolates exhibited only minor variation in their resistance to steam and lactic acid treatments but were more resistant than reference strains (three strains) or isolates from live cattle (seven strains). D₆₀ values of strains from live cattle, and reference strains ranged from 0·1 to 0·5 min, in keeping with literature data. However, D₆₀ values of current slaughter plant isolates ranged between 15 for E. coli DM18.3 and 71 min AW 1.7. Cell counts of E. coli AW 1.7 were reduced by <5 log₁₀ CFU g^?1 in ground beef patties cooked to an internal temperature of 71°C. Conclusions:  Strains of E. coli that survive cooking of ground beef to the recommended internal temperature of 71°C can be isolated from beef‐processing facilities. Significance and Impact of the Study:  Pathogen interventions in current commercial beef slaughter may select for extremely heat‐resistant strains of E. coli.     

Screening ofBifidobacterium strains for bacteriocin production

Summary Thirteen strains of bifidobacteria (Bifidobacterium) were examined for bacteriocin production. One strain among these produced bacteriocin which is active against lactic streptococci strains and against other species ofBifidobacterium, Clostridium andLactobacillus, but not against Gram-negative bacteria such asPseudomonas, Klebsiella, Serratia andEscherichia coli.Bacteriocin activity was inhibited by proteolytic enzymes, but not by heating 15 min or30 min at 100°C, and it was resistant for 15 min at 121°C and active at pH between 2 to 10.     

The fungicidal efficacy of various commercial disinfectants used in the food industry

The antifungal effects of eight commercial disinfectants namely alcohol, peracetic acid, iodophors, aldehydes, quaternary amine compounds (QAC, a, b and c), and a chlorine-based agent were assessed at different concentrations. The time taken for these disinfectants to kill different microorganisms was used to assess their efficacy. The microorganisms tested were six yeasts,Saccharomyces cerevisiae, Saccharomyces uvarum, Kloeckera apiculata, Candida oleophila, Metschnikowia fructicola, Schizosaccharomyces pombe, and two moulds,Aspergillus niger (5 strains) andPenicillium roqueforti (5 strains). The disinfectants QAC (a) and QAC (c) were the most effective against all the microorganisms tested. The chlorine-based disinfectant worked most efficiently against the moulds at all concentrations used (0.5, 1.0, 1.5 and 2.0%). Peracetic acid and alcohol based disinfectants were most effective against the yeasts than mould. Tested yeasts were more resistant to the aldehyde and iodophors base disinfectants than the others.     

Type 1 fimbriation is negatively regulated by cyclic AMP and its receptor proteinvia conjugative plasmid F inEscherichia coli K-12

Expression of fimbriation was studied inEscherichia coli K-12 CA8000 HfrH, and itscya, crp and MS2 resistant mutants. The cells of cya⁺ crp⁺ parent strain were observed to be flagellated bacilli, lacking fimbriae, unable to agglutinate erythrocytes and deficient in ability to produce surface pellicle during growth in stationary culture. The cells ofcya andcrp mutants were observed to be cocci or coccobacilli devoid of flagella, having haemagglutinating activity, fimbriated and capable of producing surface pellicle in stationary cultures. The fimbriation and haemagglutinating activities were lower incya mutants grown with cAMP supplementation. Thecya andcrp mutants produced relatively small, smooth and compact colonies consisting mostly of fimbriated cells, like those of earlier described Fimσ mutants. Thecya ⁺ crp⁺ MS2 resistant mutant produced large sized colonies like those of parent but was deficient in conjugal donor ability. It resembledcya andcrp mutants in haemagglutinating and fimbriation properties. Thecya andcrp mutants have been earlier shown to be deficient in several Tra functions including conjugal donor ability. It is concluded thatEscherichia coli K-12 cells express fimbriation when Tra functions of F-plasmid carried in them are not expressed either due to deficiency of active cAMP-receptor protein complex or mutation in F-plasmid or when F-plasmid is absent.     

Application of biotinylated and32P probes for detection of P-fimbriae in urinaryE. coli

Escherichia coli is the common causative agent of urinary tract infections. Twenty-six strains ofEscherichia coli were isolated from children with pyelonephritis, symptomatic urinary tract infections and asymptomatic bacteriuria. Biotinylated and³²P-DNA probes were prepared for detection of P-fimbriae in the isolates. Of the 13 strains isolated from patients with pyelonephritis 11 were positive for the presence of the P gene by both probes. Strains isolated from cases of symptomatic urinary tract infections revealed the presence of P gene only in three samples of the total of nine isolated. None of the isolatedE. coli strains from asymptomatic bacteriuria was found positive for the presence of the P gene. The biotynylated probe was simple and easily applicable in standard laboratory conditions and therefore the authors recommend it for use in diagnostic laboratories.