non-O157 Strains Research Articles

Evaluation of the biocide properties of whey-protein edible films with potassium sorbate to control non-O157 shiga toxinproducing Escherichia coli

The use of edible films to release antimicrobial constituents in food packaging is a form of active packaging that contributes to extend the shelf-life of a product and provides microbial safety for consumers. A number of plant and animal proteins have been investigated for the production of edible films such as corn zein, wheat gluten, soy and peanut proteins, gelatin, collagen, casein, and whey proteins. Several antimicrobial agents such as organic acids, enzymes, fungicides and natural antimicrobial compounds (spices and essential oils) can be incorporated into edible films. Potassium sorbate (PS) have a long history as a generally recognized as safe food preservative, being widely used to inhibit or retard the growing of a number of recognized food pathogens. Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 strains have been associated with human disease, ranging from uncomplicated diarrhea to hemorrhagic colitis and hemolytic uremic syndrome. STEC is transmitted to humans through contaminated food, water, and direct contact with infected persons or animals. Several outbreaks caused by non-O157 STEC were described although data implicating these STEC were scanty and the source of infection was not always known. Therefore, the objective of the present study was to incorporate PS into whey protein concentrate (WPC) films and to determine the inhibitory effects of these films against eight non-O157 STEC strains isolated from readyto-eat food samples.

Comparative PCR-based fingerprinting of Vibrio cholerae isolated in Malaysia

Vibrio cholerae, the causative agent of cholera, is endemic in many parts of the world, especially in countries poor in resources. Molecular subtyping of V. cholerae is useful to trace the regional spread of a clone or multidrug-resistant strains during outbreaks of cholera. Current available PCR-based fingerprinting methods such as Random Amplified Polymorphic DNA (RAPD)-PCR, Enterobacterial Repetitive Intergenic Consensus Sequence (ERIC)-PCR, and Repetitive Extragenic Palindromic (REP)-PCR were used to subtype V. cholerae. However, there are problems for inter-laboratory comparison as these PCR methods have their own limitations especially when different PCR methods have been used for molecular typing. In this study, a Vibrio cholerae Repeats-PCR (VCR-PCR) approach which targets the genetic polymorphism of the integron island of Vibrios was used and compared with other PCR-based fingerprinting methods in subtyping. Forty-three V. cholerae of different serogroups from various sources were tested. The PCR-fingerprinting approaches were evaluated on typeability, reproducibility, stability and discriminatory power. Overall, Malaysian non-O1/non-O139 V. cholerae were more diverse than O1 strains. Four non-O1/non-O139 strains were closely related with O1 strains. The O139 strain in this study shared similarity with strains of both O1 and non-O1/non-O139 serogroups. ERIC-PCR was the most discriminative approach (D value = 0.996). VCR-PCR was useful in discriminating non-O1/non-O139 strains. RAPD-PCR and REP-PCR were less suitable for efficient subtyping purposes as they were not reproducible and lacked stability. The combination of the ERIC-PCR and VCR-PCR may overcome the inadequacy of any one approach and hence provide more informative data.

Occurrence of Vibrio cholerae non-O1 in three wastewater treatment plants in Agadir (Morocco)

A total of 21 isolates of Vibriocholerae non-O1 strains were isolated from three wastewater treatment plants in Agadir, Morocco. The isolates were analyzed by biochemical analysis, antibiogram, pulsed-field gel electrophoresis and the MALDI-TOF patterns of their protein masses were compared. Over 67% of isolates were susceptible to antimicrobial agents tested and 14% proved resistant to both trimethoprim-sulfamethoxazole and nalidixic acid. Typing by pulsed-field gel electrophoresis with NotI digestion revealed that the V. cholerae non-O1 strains from Agadir (Morocco) have a lower level of genetic homogeneity, the restriction patterns of whole-chromosomal DNA grouped the V. cholerae O1 and V. alginolyticus strains into a separate cluster from V. metschnikovii and V. cholerae non-O1 isolates. Furthermore, to gain additional analytical accuracy and reliability in the analysis we used dendrogram based on MALDI-TOF spectral patterns generated by the BioTyper 1.1™ software. All m/z signatures of all strains tested indicate that the mass spectral data contained sufficient information to distinguish between strains of V. cholerae.

Antimicrobial Resistance Testing of Verocytotoxin-Producing Escherichia coli and First Description of TEM-52 Extended-Spectrum β-Lactamase in Serogroup O26

We have investigated the antimicrobial resistance of verocytotoxin-producing Escherichia coli (VTEC) strains isolated from humans, animals, food, and the environment in Belgium. Resistance was more frequent in non-O157 strains from humans than in O157 strains from humans or other sources, and among non-O157 VTEC strains, intimin-positive strains were more resistant than intimin-negative strains. We also report the first VTEC strain producing an IncI1 extended-spectrum β-lactamase encoded by plasmid-borne bla(TEM-52); this β-lactamase was previously associated with Salmonella enterica and E. coli isolates from different origins.

Comparison of Shiga Toxin-Producing Escherichia coli Detection Methods Using Clinical Stool Samples

Molecular diagnostic tools capable of identifying Shiga toxin-specific genetic determinants in stool specimens permit an unbiased approach to detect Shiga toxin-producing Escherichia coli (STEC) in clinical samples and can indicate when culture-based isolation methods are required. It is increasingly recognized that clinically relevant STEC are not limited to the singular O157 serotypes, and therefore diagnostic assays targeting toxin-encoding determinants must be able to account for any genetic variation that exists between serotypes. In this study conventional PCR and four real-time PCR assays (HybProbe, TaqMan, SYBR Green, and LUX) targeting the stx1 and stx2 Shiga toxin coding sequences were used to identify STEC in enriched stool samples (n = 36) and a panel of O157 and non-O157 strains (n = 64). PCR assays targeting stx1 and stx2 had variable specificity and sensitivity values with enriched stool samples. Molecular assays using DNA from pure cultures revealed that some primers were not sensitive to all stx2 variants. This evaluation concluded that the TaqMan-based probes were most appropriate in high throughput clinical diagnostic laboratories in consideration of cost, turn around time, and assay performance.

Phylogenetic analysis and Shiga toxin production profiling of Shiga toxin-producing/enterohemorrhagic Escherichia coli clinical isolates

Shiga toxin-producing Escherichia coli (STEC) can cause severe illnesses in humans such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, we carried out genotypic analysis of the Shiga toxin ( stx) gene in 120 clinical isolates of STEC and enterohemorrhagic E. coli (EHEC) from patients in a southern district of Japan. We identified 88 stx 1 + and 103 stx 2 + strains. We further identified 12 stx 1 + and stx 2 + isolates expressing little or no Shiga toxin 1 (Stx 1) and/or 2 (Stx 2) by reversed passive latex agglutination (RPLA) and Vero cell toxicity assays. Among them, 1 strain could not produce Stx 1, 8 could not produce Stx 2, and 3 strains could produce neither. Two of the latter three strains were of the non-O157 serotype. Most of the Stx RPLA-negative strains belonged to the stx 1 / stx 2 subtype (11/12, [91.7%]). Our quantitative reverse transcription PCR analysis indicated that the stx genes were not effectively transcribed in the RPLA-negative strains. This is the first report of the isolation of stx-positive strains showing Stx-negative phenotype from stx 1 -bearing strains and non-O157 strains.

Occurrence and composition of class 1 and class 2 integrons in clinical and environmental O1 and non-O1/non-O139 Vibrio cholerae strains from the Brazilian Amazon

This study identified and characterised class 1 and 2 integrons in clinical and environmental Vibrio cholerae O1 and non-O1/non-O139 strains isolated from the Brazilian Amazon. The aadA2 and aadA7 gene cassettes were found in class 1 integrons in two genotypes of environmental V. cholerae non-O1/non-O139. Empty integrons were found in strains from the Brazilian cholera epidemic. A class 2 integron was detected in one strain from the V. cholerae Amazonia lineage harbouring sat1 and aadA1 genes. All isolates were resistant to aminoglycosides, indicating aadA functionality. These findings suggest that environmental bacteria act as cassette reservoirs that favour the emergence of resistant pathogens.

Diversity and distribution of cholix toxin, a novel ADP‐ribosylating factor from Vibrio cholerae

Non-toxigenic non-O1, non-O139 Vibrio cholerae strains isolated from both environmental and clinical settings carry a suite of virulence factors aside from cholera toxin. Among V. cholerae strains isolated from coastal waters of southern California, this includes cholix toxin, an ADP-ribosylating factor that is capable of halting protein synthesis in eukaryotic cells. The prevalence of the gene encoding cholix toxin, chxA, was assessed among a collection of 155 diverse V. cholerae strains originating from both clinical and environmental settings in Bangladesh and Mexico and other countries around the globe. The chxA gene was present in 47% of 83 non-O1, non-O139 strains and 16% of 72 O1/O139 strains screened as part of this study. A total of 86 chxA gene sequences were obtained, and phylogenetic analysis revealed that they fall into two distinct clades. These two clades were also observed in the phylogenies of several housekeeping genes, suggesting that the divergence observed in chxA extends to other regions of the V. cholerae genome, and most likely has arisen from vertical descent rather than horizontal transfer. Our results clearly indicate that ChxA is a major toxin of V. cholerae with a worldwide distribution that is preferentially associated with non-pandemic strains.

Occurrence of theVibrio choleraeSeventh Pandemic VSP-I Island and a New Variant

Using comparative genomics, we identified a new variant of the Vibrio Seventh Pandemic Island-I (VSP-I). Results of polymerase chain reaction (PCR) screening for both known VSP-I variants indicate that the novel variant is present only in non-O1/non-O139 strains of V. cholerae and Vibrio mimicus. Comparative genomics revealed little sequence divergence in the seventh pandemic VSP-I; however, a second insertion site located on the smaller chromosome was identified. Although the seventh pandemic VSP-I genomic island was detected in all seventh pandemic V. cholerae serogroup O1 and O139 isolates examined in this study, unique genes of the island cannot be used alone as an identifying target, because the seventh pandemic VSP-I was also present in three non-seventh pandemic strains of V. cholerae isolated from Chesapeake Bay. As an alternative, a PCR assay targeting the VC2346 gene was found to be confirmatory for seventh pandemic isolates of V. cholerae.

Detection, isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) in fresh ground beef from butcher shops in Concepción, Tucumán Province

Shiga toxin-producing Escherichia coli is an emerging foodborne pathogen. There are many STEC serotypes associated with human diseases, being the O157:H7 serotype the most prevalent. Ground beef is the main transmission vehicle. In Concepción city, Tucumán Province, between September and December 2004, two hemolytic uremic syndrome (HUS) cases were diagnosed. The main objective of this work was to detect, isolate and characterize STEC O157 and non-O157 strains in fresh ground beef. Between September and December 2004, 53 fresh ground beef samples were collected from butcher shops in Concepción city. The USDA-FSIS (2002) methodology was used for detection, isolation and characterization of STEC O157:H7. Two PCR techniques for E. coli non-O157 detection and a previous intra-laboratory validated methodology for the isolation and characterization of these strains were used. The stx2 gen was identified in seven samples and the rfbO157 gene also in four of them. However, only one E. coli O157:H7 strain, biotype C, carrying the eae, stx2 and ehxA genes, was isolated. The present study shows the importance of implementing techniques for the detection of this emerging pathogen in meat samples.

Ecological Study of Vibrio cholerae in Aquatic Environments, Okayama

An ecological and molecular-epidemiological study of Vibrio cholerae in some aquatic environments of Okayama was carried out. The strains of non-O1/non-O139 were isolated frequently, and unconventional O1 strains were rarely observed. These non-O1/non-O139 strains did not have ctxA, the gene of choleratoxin, the major pathogenic factor of epidemic cholera, but possessed hlyA, a gene encoding hemolysin thought to be a pathogenic factor for sporadic diarrhea or food poisoning. Furthermore these strains also had toxR, a gene controlling the pathogenic island of the V. cholerae genome, suggesting the potentia of these strains for accepting the horizontal transfer of virulence factor genes. Thus, continuous survey of the vibrio is to ensure the food safety of fishery products.

Characterization of potentially virulent non-O1/non-O139 Vibrio cholerae strains isolated from human patients

Traditional methods of typing Vibrio cholerae define virulent strains according to their recognition by sera directed against the known epidemic serogroups O1 and O139, overlooking potentially virulent non-O1/non-O139 strains. Here, we have undertaken the characterization of eight clinical isolates of non-O1/non-O139 V. cholerae, collected during cholera outbreaks in Brazil. Seven of these were typed as O26 and one, 17155, was defined as non-typable. A PCR-based approach has previously detected in these strains several virulence genes derived from the CTXφ prophage and generally associated with pathogenic strains. Here, the presence of the O1-specific wbeN gene was investigated through PCR and found to be restricted to strain 17155, as well as one of the O26 strains, 4756, although neither strain was recognized by O1-specific antisera. The same two isolates were the only strains able to express the cholera toxin in culture, assayed by western blotting. They also possessed four repeats of the heptanucleotide TTTTGAT upstream of the ctxAB genes encoding the cholera toxin. The remaining strains possessed only two intact repeats, whereas pathogenic O1 possessed four to six repeats. To define their evolutionary relationships, selected 16S–23S intergenic rRNA spacer regions were sequenced from the various strains and the resulting sequences used to build phylogenetic trees. Strains 4756 and 17155 always clustered with control O1 strains, whereas the remaining O26 strains clustered separately. These results confirm that, despite their serological phenotype, these two strains are genotypically related to O1 strains and potentially able to produce epidemic cholera.

VopF, a type III effector protein from a non-O1, non-O139 Vibrio cholerae strain, demonstrates toxicity in a Saccharomyces cerevisiae model

VopF, a type III effector protein, has been identified as a contributory factor to the intestinal colonization of type III secretion system-positive, non-O1, non-O139 Vibrio cholerae strains. To gain more insight into the function of VopF, a yeast model was developed. Using this model, it was found that ectopic expression of VopF conferred toxicity in yeast.

Characterisation of verotoxin-producing Escherichia coli strains isolated from human patients in Hungary over a 7-year period

The purpose of this study was to characterise verotoxigenic Escherichia coli (VTEC) strains isolated in Hungary from 2000 to 2006. Altogether, 33 human VTEC strains were investigated to define the O:H antigens, verotoxin 1, 2 (vtx1 and 2), intimin (eae), enteroaggregative heat-stable toxin (ast1), autoagglutinating adhesin (saa) and enterohaemolysin (ehlyA) genes and sensitivity to 11 antimicrobial agents. The strains belonged to 14 different O:H serotypes, among which O157:NM (non-motile) was the most prevalent (45%, 15/33). Patients infected with O157 more often presented bloody diarrhoea or haemorrhagic colitis (63%, 12/19) than those infected with non-O157 (46%, 6/14). Haemolytic uraemic syndrome evolved in two patients infected with O26:H11. The vtx1vtx2c toxin gene combination was found in 58% (11/19) and vtx2c alone in 31% (6/19) of the O157 strains. All of the O157 strains possessed gamma1, while two O26 strains had the beta1 intimin gene. Twenty strains (75%, 25/33) carried the ehlyA gene and five non-O157 strains had ast1. The majority of the strains (76%) were resistant to at least one antimicrobial agent, but none of them showed the extended-spectrum beta-lactamase (ESBL) phenotype.

Successful Small Intestine Colonization of Adult Mice by Vibrio cholerae Requires Ketamine Anesthesia and Accessory Toxins

Vibrio cholerae colonizes the small intestine of adult C57BL/6 mice. In this study, the physical and genetic parameters that facilitate this colonization were investigated. Successful colonization was found to depend upon anesthesia with ketamine-xylazine and neutralization of stomach acid with sodium bicarbonate, but not streptomycin treatment. A variety of common mouse strains were colonized by O1, O139, and non-O1/non-O139 strains. All combinations of mutants in the genes for hemolysin, the multifunctional, autoprocessing RTX toxin (MARTX), and hemagglutinin/protease were assessed, and it was found that hemolysin and MARTX are each sufficient for colonization after a low dose infection. Overall, this study suggests that, after intragastric inoculation, V. cholerae encounters barriers to infection including an acidic environment and an immediate immune response that is circumvented by sodium bicarbonate and the anti-inflammatory effects of ketamine-xylazine. After initial adherence in the small intestine, the bacteria are subjected to additional clearance mechanisms that are evaded by the independent toxic action of hemolysin or MARTX. Once colonization is established, it is suggested that, in humans, these now persisting bacteria initiate synthesis of the major virulence factors to cause cholera disease. This adult mouse model of intestinal V. cholerae infection, now well-characterized and fully optimized, should serve as a valuable tool for studies of pathogenesis and testing vaccine efficacy.

Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.

Presence of dfr6 Gene Cassette in Superintegron of Non-O1/Non-O139 Strain of Vibrio cholerae

Integrons, the DNA elements capable of capturing small mobile elements or gene cassettes, play a major role in the spread of antibiotic resistance in gram-negative bacteria. They are generally divided into two major groups: the multiresistance integrons (MRI), which carry mobile genetic elements, and the chromosomal superintegrons (SI). Gene cassettes carried by MRI encode resistance against antibiotics and are located in either chromosomes or plasmids. Based on the divergence of integrase genes, five classes of MRI have been reported (6). The SI identified in the genomes of the pathogenic Vibrio species sequenced to date have been found to contain a large number of gene cassettes, ranging from 72 to more than 200 (3, 7). The cassettes of the Vibrio cholerae SI were demonstrated to be substrates for the class 1 integrons of MRI (5, 8). Two class 1 integron gene cassettes, CARB4 and dfr6, were found to contain attC sites similar to those of V. cholerae repeats (VCRs) (5). Hence, it was predicted that such genes were recruited from SI. The strain identified (V. cholerae non-O1/non-O139, A444) was isolated from Vembanad Lake in the Allapuzha district of Kerala, India. The antimicrobial susceptibility test was done by using commercially available discs (Himedia). The presence of the SXT element and class 1 integrons was tested by PCR as described previously (1, 9). Shotgun cloning was performed by digesting genomic DNA with BfuC1 (NEB) and ligating the fragments with the BamHI (NEB)-digested pUC19 vector. The chemically competent Escherichia coli JM109 cells were transformed with a ligation mixture. The transformants were selected at 37°C on LB agar with ampicillin (100 mg/liter) and trimethoprim (TMP) (50 mg/liter). The plasmids from TMP-resistant clones were isolated and sequenced using a pUC/M13 universal pair of primers specific for the pUC series of vector. The sequencing was performed with an ABI Prism BigDye terminator kit using an ABI Prism 3100 DNA sequencer (Applied Biosystems). The nucleotide and deduced protein sequences were analyzed with BioEdit software (version 7.0.9.0; T. Hall, http://www.mbio.ncsu.edu/BioEdit/bioedit.html) and the BLAST search engine. V. cholerae strain A444 was resistant to ampicillin, polymixin B, TMP, cotrimoxazole, streptomycin, and furazolidone. The SXT element and class 1 integrons were shown to carry several genes encoding resistance to TMP, sulfamethoxazole, streptomycin, and spectinomycin in clinical and environmental isolates of V. cholerae (2, 9), but neither the element nor the integrons could be detected in A444. The sequencing analysis revealed that the size of the insert in the pUC19 vector is 1,708 bp. The BLAST analysis of the sequence showed the presence of intIA, dfr6, hypothetical proteins, and VCR. The dfr6 gene and one of the hypothetical proteins were flanked by VCR, and the arrangement resembled typical SI structure. The dfr6 (474 bp) obtained showed 96% similarity to the dfr6 gene reported from the class 1 integron in V. cholerae O1 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB200915,term_id:58219327,term_text:AB200915}}AB200915). Further, internal primers designed to amplify a 1,062-bp region from the cloned fragment in the pUC19 vector yielded a similar amplicon when PCR was done using genomic DNA as a template. The location of cassettes encoding hypothetical proteins was found within the SI of V. cholerae El Tor strain N16961 chromosome 2. Recently, novel dfr gene cassettes in the chromosomal integrons of environmental Vibrio splendidus were identified (4). Thus, the gene cassettes of SI in the Vibrionaceae family may constitute a reservoir of antibiotic resistance genes, such as dfr6, blaCARB-7, and blaCARB-9. To our knowledge, this is the first report of a demonstration of the dfr6 gene in SI of V. cholerae. Further studies are required to identify other gene cassettes in SI from non-O1/non-O139 strains of V. cholerae. This study provides evidence to support the in vivo capture of VCR cassettes by class 1 integrons.

Hemolytic uremic syndrome (HUS)—incidence and etiologies at a regional Children’s Hospital in 2001–2006

Hemolytic uremic syndrome (HUS) is a serious health concern in children. HUS has primarily been linked to Escherichia coli O157:H7 infections, but non-O157 strains are gaining attention. Hemolytic anemia, thrombocytopenia, and acute renal failure are the characteristics of the syndrome. This study investigated the incidence of HUS at a regional Children's Hospital between 2001 and 2006 by retrospective review. Cases of HUS were investigated for outcomes based on stool culture and an association of acute pancreatitis. A total of 44 cases were identified, of which 57% were female and 43% were male, with an age distribution of 13 months to 17 years and a median age of 3.44 years. Data revealed 13 cases in 2006 compared to two cases in 2001, with 84% of all illnesses occurring in the summer and fall seasons. The median duration of thrombocytopenia was eight days and 50% of all cases required dialysis. E. coli O157:H7 was the predominant pathogen; however, 53% of the cases had unknown etiology. This data may suggest a growing number of cases from 2001 to 2006 and a role for agents other than E. coli O157:H7. E. coli O157:H7 caused longer intensive care unit (ICU) stay. No association between HUS and acute pancreatitis was found.

Changes in cell surface properties of shiga toxigenic Escherichia coli by Quercus infectoria G. Olivier.

The effects of Quercus infectoria (family Fagaceae) nutgalls on cell surface properties of Shiga toxigenic Escherichia coli (STEC) were investigated with an assay of microbial adhesion to hydrocarbon. The surface of bacterial cells treated with Q. infectoria exhibited a higher level of cell surface hydrophobicity (CSH) toward toluene than did the surface of untreated cells. With 50% ethanolic extract, the CSH of the three strains of STEC O157:H7 treated with 4X MIC of the extract resulted in moderate or strong hydrophobicity, whereas at 2x MIC and MIC, the CSH of only one strain of E. coli O157:H7 was significantly affected. The 95% ethanolic extract had a significant effect on CSH of all three strains at both 4X MIC and 2X MIC but not at the MIC. The effect on bacterial CSH was less pronounced with the other STEC strains. At 4X MIC, the 50% ethanolic extract increased the CSH of all non-O157 STEC strains significantly. At 2X MIC and 4X MIC, the 95% ethanolic extract affected the CSH of E. coli O26:H11 significantly but did not affect E. coli O111 :NM or E. coli O22. Electro microscopic examination revealed the loss of pili in the treated cells. The ability of Q. infectoria extract to modify hydrophobic domains enables this extract to partition the lipids of the bacterial cell membrane, rendering the membrane more permeable and allowing leakage of ions and other cell contents, which leads to cell death. Further studies are required to evaluate the effects of Q. infectoria extract in food systems or in vivo and provide support for the use of this extract as a food additive for control of these STEC pathogens.

Potential Applicability of Chymotrypsin-Susceptible Microcin J25 Derivatives to Food Preservation

Microcin J25 (MccJ25) is a 21-residue ribosomally synthesized lariat peptide antibiotic. MccJ25 is active against such food-borne disease-causing pathogens as Salmonella spp., Shigella spp., and Escherichia coli, including E. coli O157:H7 and non-O157 strains. MccJ25 is highly resistant to digestion by proteolytic enzymes present in the stomach and intestinal contents. MccJ25 would therefore remain active in the gastrointestinal tract, affecting normal intestinal microbiota, and this limits the potential use of MccJ25 as a food preservative. In the present paper, we describe a chymotrypsin-susceptible MccJ25 derivative with a mutation of Gly(12) to Tyr that retained almost full antibiotic activity and efficiently inhibited the growth of pathogenic Salmonella enterica serovar Newport and Escherichia coli O157:H7 in skim milk and egg yolk. However, unlike the wild-type MccJ25, the MccJ25(G12Y) variant was inactivated by digestive enzymes both in vitro and in vivo. To our knowledge, our results represent the first example of a rational modification of a microcin aimed at increasing its potential use in food preservation.