The influence of nonconjugative Escherichia coli plasmids on biofilm formation and resistance

Aims: This work describes the effects of the presence of nonconjugative plasmids in Escherichia coli cells forming biofilms on a flow cell system under turbulent conditions. Methods and Results: The pET28 and pUC8 plasmids were separately used to transform E. coli JM109(DE3). Biofilm formation, removal and antimicrobial susceptibility to the cationic biocide benzyldimethyldodecylammonium chloride (BDMDAC) were assessed. Transformed cells formed thicker biofilms with higher cell densities, and the metabolic activity was higher whereas nontransformed cells had higher viabilities. Biocide treatment was not efficient for biofilm removal but was effective for cell killing. Biofilms formed by nontransformed cells were less affected by the treatment. Conclusions: Cell transformation with the tested plasmids has significant impacts on biofilm formation, cell viability, metabolic activity and resistance to biocide treatment. Our results show that in biofilm studies involving deletion/complementation experiments, a control with the strain carrying a plasmid devoid of the gene under investigation must be included so that the real effects of the genetic manipulation are not biased by the presence of the plasmid backbone. Significance and Impact of the Study: This is the first report where the presence of nonconjugative plasmids is assessed in flow conditions analysing biofilm formation, removal and antimicrobial susceptibility of high cell‐density biofilms.     

Plasmid transfer by conjugation from Escherichia coli to Gram-positive bacteria

Abstract We have developed a vector strategy that allows transfer of plasmid DNA by conjugation from Escherichia coli to various Gram-positive bacteria in which transformation via natural competence has not been demonstrated. The prototype vector constructed, pAT187, contains the origins of replication of pBR322 and of the broad host range streptococcal plasmid pAMβ1, a kanamycin resistance gene known to be expressed in both Gram-negative and Gram-positive bacteria, and the origin of transfer of the IncP plasmid RK2. This shuttle plasmid can be mobilised efficiently by the self-transferable IncP plasmid pRK212.1 co-resident in the E. coli donors, and was successfully transferred by filter matings at frequencies of 2 × 10⁻⁸ to 5 × 10⁻⁷ to Enterococcus faecalis, Streptococcus lactis, Streptococcus agalactiae, Bacillus thuringiensis, Listeria monocytogenes and Staphylococcus aureus .     

Plasmid retention and gene expression in suspended and biofilm cultures of recombinant Escherichia coli DH5alpha(pMJR1750)

Differences in plasmid retention and expression are studied in both suspended and biofilm cultures of Escherichia coli DH5alpha(PMJR1750). An alternative mathematical model is proposed which allows the determination of plasmid loss probability in both suspended batch and continuously fed biofilm cultures. In our experiments, the average probability of plasmid loss of E. coli DH5alpha(pMJR1750) is 0.0022 in batch culture in the absence of antibiotic selection pressure and inducer. Under the induction of 0.17 MM IPTG, the maximum growth rate of plasmid-bearing cells in suspended batch culture dropped from 0.45 h(-1) to 0.35 h(-1) and the beta-galactosidase concentration reached an experimental maximum of 0.32. pg/cell 4 hours after the initiation of induction. At both 0.34 and 0.51 mM IPTG, growth rates in batch cultures decreased to 0.16 h(-1), about 36% of that without IPTG, and the beta-galactosidase concentration reached an experimental maximum of 0.47 pg/cell 3 hours after induction.In biofilm cultures, both plasmid-bearing and plasmid-free cells in increase with time reaching a plateau after 96 hours n the absence of both the inducer and any antibiotic selection pressure. Average probability of plasmid loss for biofilm-bound E. coli DH5beta(pMJR1750) population was 0.017 without antibiotic selection. Once the inducer IPTG was added, the concentration of plasmid-bearing cells in biofilm dropped dramatically while plasmid-free cell numbers maintained unaffected. The beta-galactosidase concentration reached a maximum in all biofilm experiments 24 hours after induction; they were 0.08, 0.1, and 0.12 pg/cel under 0.17, 0.34, and 0.51 mM IPTG, respectively. (c) 1993 John Wiley & Sons, Inc.     

Microwave improved Escherichia coli transformation

Aims: The calcium chloride chemical transformation of Escherichia coli is still the most widley used cloning method in small laboratories. Therefore, any practicable improvement in its transformation efficiency seems to be of general interest. Methods and Results: We found that giving calcium chloride competent cells a 1 min microwave pulse at the lowest power setting (180 W), instead of the classic 1–2 min 42°C heat‐shock step, increases the transformation efficiency around threefold (3.3 ± 0.5). Moreover, when both treatments were given in a 2‐min 42°C ? 5 min on ice ? 1 min microwave pluse sequence, an additional improvement of 1.6 was obtained, resulting in an overall increase in efficiency of approximately 5.3‐fold compared to classical heat shock. Conclusions: This transformation method significantly improves the classical heat shock treatment. Significance and Impact of the Study: This method might be useful to those laboratories that cannot afford an electroporation apparatus.     

外源基因在大肠杆菌中的高效表达

为了提高外源蛋白在大杨杆菌中的表达量,人们对大肠杆菌表达系统进行了许多研究。作者综述了有关外源基因在大肠杆菌中高效表达的研究进展。     

Horizontal gene transfer of a genetic island encoding a type III secretion system distributed in Vibrio cholerae

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS‐related genes were distributed among the various serogroups and pulsed‐field gel electrophoresis of NotI‐digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS‐related genes had similar patterns. Additionally, naturally competent T3SS‐negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS‐related genes occurs among V. cholerae in natural ecosystems.     

Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent

Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays.
Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm.
Conclusions:  Escherichia coli forms biofilms on static assays in a method-dependent fashion, depending on strain, and it is strongly modulated by culture conditions.
Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.     

Isolation of a conjugative plasmid in Escherichia coli determining formaldehyde resistance

A clinical isolate of Escherichia coli which was resistant to the disinfectant formaldehyde was investigated. The strain harboured a plasmid of 62 MDa size. It was shown by conjugation, transformation and plasmid-curing experiments that the formaldehyde resistance is plasmid-mediated and transferable to other strains.     

Effect of carbon source addition on toluene biodegradation by an Escherichia coli DH5α transconjugant harboring the TOL plasmid

Horizontal gene transfer (HGT) of plasmids is a naturally occurring phenomenon which could be manipulated for bioremediation applications. Specifically, HGT may prove useful to enhance bioremediation through genetic bioaugmentation. However, because the transfer of a plasmid between donor and recipient cells does not always result in useful functional phenotypes, the conditions under which HGT events result in enhanced degradative capabilities must first be elucidated. The objective of this study was to determine if the addition of alternate carbon substrates could improve toluene degradation in Escherichia coli DH5α transconjugants. The addition of glucose (0.5–5 g/L) and Luria–Bertani (LB) broth (10–100%) resulted in enhanced toluene degradation. On average, the toluene degradation rate increased 14.1 (±2.1)‐fold in the presence of glucose while the maximum increase was 18.4 (±1.7)‐fold in the presence of 25% LB broth. Gene expression of xyl genes was upregulated in the presence of glucose but not LB broth, which implies different inducing mechanisms by the two types of alternate carbon source. The increased toluene degradation by the addition of glucose or LB broth was persistent over the short‐term, suggesting the pulse amendment of an alternative carbon source may be helpful in bioremediation. While the effects of recipient genome GC content and other conditions must still be examined, our results suggest that changes in environmental conditions such as alternate substrate availability may significantly improve the functionality of the transferred phenotypes in HGT and therefore may be an important parameter for genetic bioaugmentation optimization. Biotechnol. Bioeng. 2010;107: 269–277. © 2010 Wiley Periodicals, Inc.     

Pathogenic potential and horizontal gene transfer in ovine gastrointestinal Escherichia coli

Aims: To demonstrate that a thorough characterization and virulotyping of Escherichia coli strains isolated from sheep over time leads to new insights into ovine E. coli potentially becoming human pathogens through horizontal gene transfer. Methods and Results: One hundred and fifty E. coli isolates from two sheep, sampled over 3 weeks, were characterized by serotyping, virulotyping, genotyping using multiple locus variable number tandem repeats analysis (MLVA) and susceptibility to phage infection in vitro. The 35 MLVA profiles and the serotype and virulotypes of the strains were closely associated. Many MLVA profiles differed in one locus independent of serotypes. Escherichia coli isolates of the same serotype or virulotype had identical or very similar MLVA profiles. No transductants that incorporated the bacteriophages were found in vivo, but six E. coli isolates were susceptible to the phage infection in vitro. Changes in MLVA profiles were seen after acquisition of Stx phages in vitro only. Conclusions: The sheep carried Stx phage susceptible E. coli that possessed virulence markers associated with human pathogenicity. Changes in bacterial genomes by phage transfer may complicate outbreak source investigations. Serotype has to be taken into account when evaluating strain relationships by MLVA. Significance and Impact of the Study: Sheep carry E. coli that encode for virulence markers and belong to serogroups known to be human pathogens. In addition, a selection of isolates was found to be susceptible to horizontal transfer of Shiga toxin genes by means of bacteriophages in vitro, and the transfer resulted in a discernible change of the MLVA patterns of E. coli.     

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as ‘Generally Recognized as Safe’ (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.     

A complete set of Escherichia coli open reading frames in mobile plasmids facilitating genetic studies.

To facilitate genetic studies of Escherichia coli, we constructed a complete set of mobile plasmid clones of intact open reading frames (ORFs). Their expression is strictly controlled by Ptac / lacI(q). The plasmids carrying each ORF were introduced into an F+ recA strain and stored in 96-well microtiter plates. In this way, 96 clones can be transferred simultaneously to F- bacteria using the conjugative system. This provides a convenient procedure for systematic identification of ORFs that suppress or complement mutations. We created two types of clone sets: the original set contained individual clones in 45 microtiter plates, and a second set contained pools of 48 clones stored in a single microtiter plate. Using these clone sets, we have identified 403 genes that can correct in trans the temperature-sensitive defect of cell division mutants, which would suggest multiple global regulators for bacterial cell division.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

Inactivation of Escherichia coli O157:H7 in biofilm on stainless steel by treatment with an alkaline cleaner and a bacteriophage

AIMS: To determine the effectiveness of an alkaline cleaner used in food-processing plants and a lytic bacteriophage specific for Escherichia coli O157:H7 in killing wild type and rpoS-deficient cells of the pathogen in a biofilm. METHODS AND RESULTS: Wild type and rpoS-deficient cells were attached to stainless steel coupons (c. 7-8 log CFU per coupon) on which biofilms were developed during incubation at 22 degrees C for 96 h in M9 minimal salts media (MSM) with one transfer to fresh medium. Coupons were treated with 100 and 25% working concentrations of a commercial alkaline cleaner (pH 11.9, with 100 microg ml(-1) free chlorine) used in the food industry, chlorine solutions (50 and 100 microg ml(-1) free chlorine), or sterile deionized water (control) at 4 degrees C for 1 and 3 min. Treatment with 100% alkaline cleaners reduced populations by 5-6 log CFU per coupon, a significant (P < or = 0.05) reduction compared with treatment with water. Initial populations (2.6 log CFU per coupon) of attached cells of both strains were reduced by 1.2 log CFU per coupon when treated with bacteriophage KH1 (7.7 log PFU ml(-1)) for up to 4 days at 4 degrees C. Biofilms containing low populations (2.7-2.8 log CFU per coupon) of wild type and rpoS-deficient cells that had developed for 24 h at 22 degrees C were not decreased by more than 1 log CFU per coupon when treated with KH1 (7.5 log PFU ml(-1)) at 4 degrees C. CONCLUSIONS: Higher numbers of cells of E. coli O157:H7 in biofilms are killed by treatment with an alkaline cleaner than with hypochlorite alone, possibly through a synergistic mechanism of alkaline pH and hypochlorite. Populations of cells attached on coupons were reduced by treating with bacteriophage but cells enmeshed in biofilms were protected. SIGNIFICANCE AND IMPACT OF THE STUDY: The alkaline pH, in combination with hypochlorite, in a commercial cleaner is responsible for killing E. coli O157:H7 in biofilms. Treatment with bacteriophage KH1 reduces populations of cells attached to coupon surfaces but not cells in biofilms.     

Lack of evidence for horizontal transfer of the lac operon into Escherichia coli

The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation.     

猪源大肠杆菌耐药质粒图谱及耐药性分析

目的:探讨猪大肠杆菌的耐药质粒图谱、耐药性及耐药基因之间的关系。方法:从湖南省株洲、益阳的四个猪场分离出9株大肠杆菌,进行质粒电泳图谱分析、用PCR法检测耐喹诺酮类耐药基因Gyr A、Par C和耐四环素类耐药基因Tet A、Tet B,并采用Kirby-bauer法对这9株大肠杆菌进行药敏(18种抗生素)试验。结果:其中9株大肠杆菌含有三条或者三条以上的质粒条带,且其质粒谱型均不相同;9株大肠杆菌均检测出4种耐药基因Gyr A、Par C、Tet A和Tet B;9株大肠杆菌对所选用的抗生素存在不同程度的耐药性,其中7株大肠杆菌对10种或10种以上的抗生素耐药,最高对13种抗生素耐药,氨苄西林、青霉素、阿莫西林、红霉素的耐药率达100%,对四环素、多西环素的耐药率达到88.9%,而多粘菌素B、阿奇霉素、大观霉素耐药率较低。结论:耐药性与质粒条带数、耐药基因之间并无明显的相关性;猪大肠杆菌呈多重耐药之势,在治疗大肠杆菌病时最好根据药敏实验结果选用合适的抗生素。     

Horizontal transfer of non-conjugative plasmid in colony biofilm of Escherichia coli on food-based media

We previously showed that non-conjugative, non-viral lateral plasmid transfer occurs in a colony biofilm of mixed Escherichia coli strains cultured on common laboratory media, such as LB agar. In this report, to investigate the possibility of this plasmid transfer under conditions possible outside the laboratory, we examined the activities of foodstuffs and mixed food extracts, which are possible nutrients for bacteria in human environments, for supporting lateral plasmid transfer. Lateral plasmid transfer occurred in colony biofilms grown on several foodstuffs (roasted meats) and on agar media containing mixed food extracts, which consisted of sugar, milk, and extracts of several foodstuffs (vegetables, fruits, and meats). Lateral plasmid transfer did not occur in liquid culture consisting of the same mixed food extracts, suggesting the importance of colony-biofilm formation. These results suggest the possibility that lateral transfer of non-conjugative plasmid between bacterial cells occurs in biofilms grown with foods or food-like nutrients in the environment.     

Characterization of typical and atypical enteroaggregative Escherichia coli in Kagoshima,Japan: biofilm formation and acid resistance

EAEC is increasingly recognized as an emerging enteric pathogen. Typical EAEC expressing the AggR regulon have been proven to be an important cause of childhood diarrhea in industrialized countries as well as in the developing world, while atypical EAEC without this regulon have not been thoroughly investigated. To investigate the bacteriological characteristics of EAEC, including both typical and atypical strains in Kagoshima, Japan, 2417 E. coli strains from Japanese children with diarrhea were screened by a quantitative biofilm assay to detect possible EAEC strains, resulting in the identification of 102 (4.2%) of these strains by the HEp‐2 cell adherence test. Virulence gene patterns, PFGE analysis and O‐serogrouping demonstrated the heterogeneity of the EAEC. The EAEC strains were classified into two groups: typical EAEC with aggR (74.5%, 76/102) and atypical EAEC without aggR (25.5%, 26/102). There was no significant difference between the typical EAEC strains (median OD₅₇₀= 0.73) and the atypical strains (median OD₅₇₀= 0.61) in biofilm formation (P= 0.17). Incidences of resistance against ampicillin, cefotaxime and tetracycline were significantly higher in the typical EAEC strains than the atypical EAEC strains (84.2% vs. 53.8%, 36.8% vs. 7.7% and 93.4% vs. 73.1%, respectively, P < 0.05). The typical EAEC strains showed significantly higher resistance ratios against HCl and lactate than the atypical strains (94.7% vs. 61.5% and 92.1% vs. 57.7%, respectively, P < 0.001). To investigate the pathogenicity of not only typical but also atypical EAEC, further bacteriological and epidemiologic studies including atypical EAEC are needed.