Diversity and Local Coadaptation of Escherichia coli and Coliphages From Small Ruminants

Bacteriophages are highly specific predators that drive bacterial diversity through coevolution while striking tradeoffs among preserving host populations for long-term exploitation and increasing their virulence, structural stability, or host range. Escherichia coli and other coliform bacteria present in the microbiota of milk and during early ripening of raw milk cheeses have been linked to the production of gas, manifested by the appearance of eyes, and the development of off-flavors; thus, they might cause early blowing and cheese spoilage. Here, we report the characterization of coliphages isolated from manure from small ruminant farms and E. coli strains isolated from goat and sheep raw milk cheese. Additionally, the virulence and host range of locally isolated and laboratory collection phages were determined by comparing the susceptibility of E. coli strains from different sources. In agreement with the high genetic diversity found within the species E. coli, clustering analysis of whole-cell protein revealed a total of 13 distinct profiles but none of the raw milk cheese isolates showed inhibition of growth by reference or water-isolated coliphages. Conversely, 10 newly isolated phages had a broad host range (i.e., able to lyse ≥50% of bacterial hosts tested), thus exhibiting utility for biocontrol and only one cheese-isolated E. coli strain was resistant to all the phages. Whereas there was a high positive correlation between bacterial susceptibility range and lysis intensity, the phages virulence decreased as range increased until reaching a plateau. These results suggest local gene-for-gene coevolution between hosts and phages with selective tradeoffs for both resistance and competitive ability of the bacteria and host-range extension and virulence of the phage populations. Hence, different phage cocktail formulations might be required when devising long-term and short-term biocontrol strategies.

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Occurrence of toxigenic Escherichia coli in raw milk cheese in Brazil

Arquivo Brasileiro de Medicina Veterinária e Zootecnia ◽

10.1590/s0102-09352007000200035 ◽

2007 ◽

Vol 59 (2) ◽

pp. 508-512 ◽

Cited By ~ 27

Author(s):

B.R. Paneto ◽

R.P. Schocken-Iturrino ◽

C. Macedo ◽

E. Santo ◽

J.M. Marin

Keyword(s):

Escherichia Coli ◽

Polymerase Chain Reaction ◽

Antimicrobial Agents ◽

Raw Milk ◽

Diffusion Method ◽

Chain Reaction ◽

Disk Diffusion Method ◽

E Coli ◽

Polymerase Chain ◽

Raw Milk Cheese

The occurrence of toxigenic Escherichia coli in raw milk cheese was surveyed in Middle Western Brazil. Fifty samples of cheese from different supermarkets were analyzed for E.coli. The isolates were serotyped and screened for the presence of verotoxigenic E. coli (VTEC) and enterotoxigenic E. coli (ETEC) by Polymerase Chain Reaction (PCR). The susceptibility to thirteen antimicrobial agents was evaluated by the disk diffusion method. E.coli were recovered from 48 (96.0%) of the samples. The serogroups identified were O125 (6.0%), O111 (4.0%), O55 (2.0%) and O119 (2.0%). Three (6.0%) and 1(2.0%) of the E.coli isolates were VTEC and ETEC, respectively. Most frequent resistance was observed to the following antimicrobials: cephalothin (60.0%), nalidixic acid (40.0%), doxycyclin (33.0%), tetracycline (31.0%) and ampicillin (29.0%).

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Comparison of the SimPlate Coliform and Escherichia coli Test with Petrifilm, Three-Tube MPN, and VRBA + MUG Methods for Enumerating Coliforms and E. coli in Food

Journal of Food Protection ◽

10.4315/0362-028x-61.4.444 ◽

1998 ◽

Vol 61 (4) ◽

pp. 444-449 ◽

Cited By ~ 10

Author(s):

D. E. TOWNSEND ◽

R. L. IRVING ◽

A. NAQUI

Keyword(s):

Escherichia Coli ◽

Correlation Coefficients ◽

Raw Milk ◽

Food Samples ◽

Coliform Bacteria ◽

Most Probable Number ◽

Additional Food ◽

Suitable Alternative ◽

Probable Number ◽

E Coli

SimPlate for coliforms and Escherichia coli (CEc) is a new method for the detection and quantification of coliforms and E. coli in food. Internal validation of the method was carried out at IDEXX Laboratories (Westbrook, ME) with 180 food samples representing a variety of different food matrices and compared against three-tube MPN (most probable number), VRBA (violet red bile agar) + MUG, and Petrifilm (E. coli count) methods. SimPlate CEc was highly correlated with each of these methods for the quantification of coliform bacteria (r ≥ 0.90). An insignificant number of food samples were found to contain E. coli; therefore, no meaningful correlation data could be generated. Four hundred forty-four additional food samples were tested at five collaborating laboratories for the presence of coliforms and E. coli using SimPlate CEc and either VRBA + MUG or Petrifilm (E. coli count). Regression analysis of data from SimPlate for CEc versus Petrifilm E. coli count plates generated correlation coefficients (r) of at least 0.89 for total coliforms and at least 0.90 for generic E. coli. Correlation coefficients between SimPlate for CEc and VRBA + MUG data were at least 0.90 for coliforms and at least 0.86 for E. coli. SimPlate for CEc demonstrated better recovery of E. coli than Petrifilm when high populations of bacteria were present. E. coli was not detected in 20 of 50 (40%) raw milk samples tested by the Petrifilm method due to the presence of interfering coliform and noncoliform bacteria. It is concluded that SimPlate for CEc is a suitable alternative for determining numbers of coliform bacteria and E. coli in food.

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LamB, OmpC, and the Core Lipopolysaccharide of Escherichia coli K-12 Function as Receptors of Bacteriophage Bp7

Journal of Virology ◽

10.1128/jvi.00325-20 ◽

2020 ◽

Vol 94 (12) ◽

Cited By ~ 1

Author(s):

Peipei Chen ◽

Huzhi Sun ◽

Huiying Ren ◽

Wenhua Liu ◽

Guimei Li ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Inner Core ◽

Phage Infection ◽

Broad Host Range ◽

Content Type ◽

E Coli ◽

Phage Adsorption ◽

Specific Receptors ◽

K 12

ABSTRACT Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions. IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38.

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Survey of Minas frescal cheese from Southwest Minas Gerais for virulence factors and antimicrobial resistance in Escherichia coli isolates

Ciência Rural ◽

10.1590/0103-8478cr20131237 ◽

2014 ◽

Vol 44 (8) ◽

pp. 1506-1511 ◽

Cited By ~ 4

Author(s):

Mõnica Hitomi Okura ◽

José Moacir Marin

Keyword(s):

Escherichia Coli ◽

Raw Milk ◽

Minas Gerais ◽

Antimicrobial Drugs ◽

E Coli ◽

Virulence Markers ◽

High Consumption ◽

Soft Cheese ◽

Minas Gerais State ◽

Raw Milk Cheese

The soft cheese Minas frescal is one of the most popular cheese in Brazil, which is typically manufactured in small dairy farms under unsatisfactory hygiene conditions. To assess the risk involved in consumption of this cheese, virulence markers were investigated in 330 Escherichia coli strains isolated from 30 Minas frescal cheeses inspected by official government agency (SIF - serviço de inspeção federal), from 50 cheeses not inspected by SIF and 31 cheeses not inspected by SIF with spice added, all of them collected in the southwest of Minas Gerais State. The E. coli isolates were screened for the presence of Shiga toxin-encoding (stx 1 and stx 2), intimin (eae) genes and for the presence of (pap, sfa, afa) genes related to adhesion in epithelial cells. The only gene detected by PCR was the sfa gene at one isolate. The strains were also screened for resistance to 9 antimicrobial drugs. Predominant resistance was to cephalothin, tetracycline and streptomycin. Multidrug resistance was found among isolates from cheese with SIF (16.6%), cheese without SIF (8.0%) and cheese without SIF with spice added (30.0%) what is a reason for concern due to the high consumption of raw milk cheese by the Brazilian population.

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Silent Mischief: Bacteriophage Mu Insertions Contaminate Products of Escherichia coli Random Mutagenesis Performed Using Suicidal Transposon Delivery Plasmids Mobilized by Broad-Host-Range RP4 Conjugative Machinery

Journal of Bacteriology ◽

10.1128/jb.00621-10 ◽

2010 ◽

Vol 192 (24) ◽

pp. 6418-6427 ◽

Cited By ~ 132

Author(s):

Lionel Ferrières ◽

Gaëlle Hémery ◽

Toan Nham ◽

Anne-Marie Guérout ◽

Didier Mazel ◽

...

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Random Mutagenesis ◽

Broad Host Range ◽

Donor Strain ◽

Bacteriophage Mu ◽

Genetic Determinants ◽

Sensitive Species ◽

E Coli ◽

Original Construction

ABSTRACT Random transposon mutagenesis is the strategy of choice for associating a phenotype with its unknown genetic determinants. It is generally performed by mobilization of a conditionally replicating vector delivering transposons to recipient cells using broad-host-range RP4 conjugative machinery carried by the donor strain. In the present study, we demonstrate that bacteriophage Mu, which was deliberately introduced during the original construction of the widely used donor strains SM10 λpir and S17-1 λpir, is silently transferred to Escherichia coli recipient cells at high frequency, both by hfr and by release of Mu particles by the donor strain. Our findings suggest that bacteriophage Mu could have contaminated many random-mutagenesis experiments performed on Mu-sensitive species with these popular donor strains, leading to potential misinterpretation of the transposon mutant phenotype and therefore perturbing analysis of mutant screens. To circumvent this problem, we precisely mapped Mu insertions in SM10 λpir and S17-1 λpir and constructed a new Mu-free donor strain, MFDpir, harboring stable hfr-deficient RP4 conjugative functions and sustaining replication of Π-dependent suicide vectors. This strain can therefore be used with most of the available transposon-delivering plasmids and should enable more efficient and easy-to-analyze mutant hunts in E. coli and other Mu-sensitive RP4 host bacteria.

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A broad-host-range event detector: expanding and quantifying performance between Escherichia coli and Pseudomonas species

Synthetic Biology ◽

10.1093/synbio/ysaa002 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 1

Author(s):

Nymul Khan ◽

Enoch Yeung ◽

Yuliya Farris ◽

Sarah J Fansler ◽

Hans C Bernstein

Keyword(s):

Escherichia Coli ◽

Host Range ◽

Memory Device ◽

Broad Host Range ◽

Complex Environments ◽

New Host ◽

E Coli ◽

Pseudomonas Fluorescens Sbw25 ◽

The Common ◽

New Applications

Abstract Modern microbial biodesign relies on the principle that well-characterized genetic parts can be reused and reconfigured for different functions. However, this paradigm has only been successful in a limited set of hosts, mostly comprised from common lab strains of Escherichia coli. It is clear that new applications such as chemical sensing and event logging in complex environments will benefit from new host chassis. This study quantitatively compared how the same chemical event logger performed across four strains and three different microbial species. An integrase-based sensor and memory device was operated by two representative soil Pseudomonads—Pseudomonas fluorescens SBW25 and Pseudomonas putida DSM 291. Quantitative comparisons were made between these two non-traditional hosts and two benchmark E. coli chassis including the probiotic Nissle 1917 and common cloning strain DH5α. The performance of sensor and memory components changed according to each host, such that a clear chassis effect was observed and quantified. These results were obtained via fluorescence from reporter proteins that were transcriptionally fused to the integrase and downstream recombinant region and via data-driven kinetic models. The Pseudomonads proved to be acceptable chassis for the operation of this event logger, which outperformed the common E. coli DH5α in many ways. This study advances an emerging frontier in synthetic biology that aims to build broad-host-range devices and understand the context by which different species can execute programmable genetic operations.

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Expanding the toolbox of broad host-range transcriptional terminators for Proteobacteria through metagenomics

10.1101/485938 ◽

2018 ◽

Author(s):

Vanesa Amarelle ◽

Ananda Sanches-Medeiros ◽

Rafael Silva-Rocha ◽

María-Eugenia Guazzaroni

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Pseudomonas Putida ◽

Host Range ◽

Broad Host Range ◽

Functional Metagenomics ◽

Alternative Hosts ◽

E Coli ◽

Broad Host Range Plasmid

AbstractAs the field of synthetic biology moves towards the utilization of novel bacterial chassis, there is a growing need for biological parts with enhanced performance in a wide number of hosts. Is not unusual that biological parts (such as promoters and terminators), initially characterized in the model bacteria Escherichia coli, do not perform well when implemented in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic circuits in industrially relevant bacteria. In order to address this limitation, we present here the mining of transcriptional terminators through functional metagenomics to identify novel parts with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas putida. Further characterization allowed the identification of 4 unique sequences between 58 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work presents a new set of biological parts useful for the engineering of synthetic circuits in Proteobacteria.

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Inhibition of Antimicrobial-Resistant Escherichia coli Using a Broad Host Range Phage Cocktail Targeting Various Bacterial Phylogenetic Groups

Frontiers in Microbiology ◽

10.3389/fmicb.2021.699630 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jinshil Kim ◽

Haejoon Park ◽

Sangryeol Ryu ◽

Byeonghwa Jeon

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Host Range ◽

Microscopic Analysis ◽

Broad Host Range ◽

Phylogenetic Groups ◽

Electron Microscopic ◽

E Coli ◽

Phage Cocktail ◽

Transmission Electron Microscopic Analysis

Antimicrobial-resistant (AMR) commensal Escherichia coli is a major reservoir that disseminates antimicrobial resistance to humans through the consumption of contaminated foods, such as retail poultry products. This study aimed to control AMR E. coli on retail chicken using a broad host range phage cocktail. Five phages (JEP1, 4, 6, 7, and 8) were isolated and used to construct a phage cocktail after testing infectivity on 67 AMR E. coli strains isolated from retail chicken. Transmission electron microscopic analysis revealed that the five phages belong to the Myoviridae family. The phage genomes had various sizes ranging from 39 to 170 kb and did not possess any genes associated with antimicrobial resistance and virulence. Interestingly, each phage exhibited different levels of infection against AMR E. coli strains depending on the bacterial phylogenetic group. A phage cocktail consisting of the five phages was able to infect AMR E. coli in various phylogenetic groups and inhibited 91.0% (61/67) of AMR E. coli strains used in this study. Furthermore, the phage cocktail was effective in inhibiting E. coli on chicken at refrigeration temperatures. The treatment of artificially contaminated raw chicken skin with the phage cocktail rapidly reduced the viable counts of AMR E. coli by approximately 3 log units within 3 h, and the reduction was maintained throughout the experiment without developing resistance to phage infection. These results suggest that phages can be used as a biocontrol agent to inhibit AMR commensal E. coli on raw chicken.

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The examination of presumed Escherichia coli count of raw milk samples on several milk production farms

Acta Agraria Debreceniensis ◽

10.34101/actaagrar/21/3170 ◽

2006 ◽

pp. 31-37

Author(s):

Ferenc Peles ◽

András Szabó ◽

Béla Béri ◽

Péter Keresztúri

Keyword(s):

Escherichia Coli ◽

Raw Milk ◽

Coliform Bacteria ◽

Farm Size ◽

Small Farms ◽

Bulk Tank Milk ◽

E Coli ◽

Significant Difference ◽

Bulk Tank ◽

Large Farms

For dairy farms, it is of great importance to insure the appropriate hygienic status of milk and to examine it regularly. Escherichia coli, belonging to the coliform bacteria type of, is a good indicator of contamination, and therefore suitable for characterising the hygienic condition of milk production.The aim of our research was to examine the connection between the Escherichi coli count in bulk tank milk and housing and milking technologies of different-sizes farms. We examined the relation using various statistical methods.Analysing the connection between the E. coli count and the farm size we found no significant difference between the farms. On the basis of the mean values of the E. coli count, we can say that the hygienic conditions are appropriate for mid-sized farms, and tolerable for large farms. We found differences in the hygienic status among the small farms. Half of the eight small farms, had no adequate hygiene. The results of the analysis of the quality categories show that the probability of inadequate quality milk was the largest on small farms (37.5%).Comparing the various housing and milking methods with each other, there were numerical differences in the E. coli count, but these differences were not significant. We got higher E. coli count values on those farms using tied stall barn and bucket milking installation. The reason for this could be that in cases of farms using bucket milking installation, it is harder to meet the requirements.After forming groups by farm size, housing and milking methods, we found that the E. coli counts are adequate on mid-size farms using various housing and milking methods; and tolerable on those large farms using loose housing stable and a milking parlour. At the same time, we found inadequate E. coli counts on the smaller farms using tied stall barns and bucket milking installation.The results show that if there is suitable attention, independent of farm size, housing and milking procedure, it is possible to produce milk with low E. coli counts, and to insure appropriate hygienic conditions.Further detailed examinations are needed to decide which factors of housing and milking technologies influence the E. coli count of bulk tank milk.

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