Genomic Diversity and Virulence Potential of ESBL- and AmpC-β-Lactamase-Producing Escherichia coli Strains From Healthy Food Animals Across Europe

The role of livestock animals as a putative source of ESBL/pAmpC E. coli for humans is a central issue of research. In a large-scale pan-European surveillance, 2,993 commensal Escherichia spp. isolates were recovered from randomly collected fecal samples of healthy cattle, pigs and chickens in various abattoirs. One-hundred Escherichia spp. isolates (0.5% from cattle, 1.3% pigs, 8.0% chickens) fulfilled the criteria for cefotaxime and ceftazidime non-wildtype (EUCAST). In silico screening of WGS data of 99 isolates (98 E. coli and 1 E. fergusonii) revealed blaSHV–12 (32.3%), blaCTX–M–1 (24.2%), and blaCMY–2 (22.2%) as predominant ESBL/pAmpC types. Other types were blaSHV–2 (1.0%), blaCTX–M–2/–14/–15 (1.0/6.1/1.0%), and blaTEM–52 (5.1%). Six isolates revealed AmpC-promoter mutations (position −42 (C > T) and one carried mcr-1. The majority (91.3%) of ESBL/pAmpC genes were located on plasmids. SHV-12 was mainly (50%) encoded on IncI1α plasmids (pST-3/-26/-95), followed by IncX3 (12.5%) and IncK2 (3.1%). The blaTEM–52 genes were located on IncI1α-pST-36 (60%) and IncX1 plasmids (20%). The dominant plasmid lineage among CTX-M-1 isolates was IncI1α (pST-3/-295/-317) (87.5%), followed by IncN-pST-1 (8.3%). CMY-2 was mostly identified on IncI1α (pST-12/-2) (54.5%) and IncK2 (31.8%) plasmids. Several plasmids revealed high similarity to published plasmids from human and animal Enterobacteriaceae. The isolates were assigned to phylogroups A/C (34.7/7.1%), B1 (27.6%), B2 (3.1%), D/F (9.2/10.2%), E (5.1%), and to E. clades (3.0%). With 51 known and 2 novel MLST types, a wide variety of STs was found, including STs previously observed in human isolates (ST10/38/117/131/648). ESBL/AmpC types or STs were rarely correlated with the geographic origin of the isolates or animal species. Virulence gene typing identified extraintestinal pathogenic E. coli (ExPEC; 2.0%), avian pathogenic E. coli (APEC; 51.5%), and atypical enteropathogenic E. coli (EPEC; 6.1%). In conclusion, the high diversity of STs and phylogenetic groups provides hardly any hint for clonal spread of single lineages but hints toward the dissemination of cephalosporin resistance genes in livestock via distinct, globally successful plasmid lineages. Even though a number of isolates could not be assigned to a distinct pathotype, our finding of combined multidrug-resistance and virulence in this facultative pathogen should be considered an additional threat to public health.

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Prevalence, Risk Factors, and Genetic Characterization of Extended-Spectrum Beta-Lactamase Escherichia coli Isolated From Healthy Pregnant Women in Madagascar

Frontiers in Microbiology ◽

10.3389/fmicb.2021.786146 ◽

2021 ◽

Vol 12 ◽

Author(s):

Milen Milenkov ◽

Saida Rasoanandrasana ◽

Lalaina Vonintsoa Rahajamanana ◽

Rivo Solo Rakotomalala ◽

Catherine Ainamalala Razafindrakoto ◽

...

Keyword(s):

Public Health ◽

Risk Factors ◽

Pregnant Women ◽

Large Scale ◽

Genomic Diversity ◽

World Health ◽

Health Concern ◽

Snp Analysis ◽

Phylogenetic Groups ◽

E Coli

Antimicrobial resistance is a major public health concern worldwide affecting humans, animals and the environment. However, data is lacking especially in developing countries. Thus, the World Health Organization developed a One-Health surveillance project called Tricycle focusing on the prevalence of ESBL-producing Escherichia coli in humans, animals, and the environment. Here we present the first results of the human community component of Tricycle in Madagascar. From July 2018 to April 2019, rectal swabs from 492 pregnant women from Antananarivo, Mahajanga, Ambatondrazaka, and Toamasina were tested for ESBL-E. coli carriage. Demographic, sociological and environmental risk factors were investigated, and E. coli isolates were characterized (antibiotic susceptibility, resistance and virulence genes, plasmids, and genomic diversity). ESBL-E. coli prevalence carriage in pregnant women was 34% varying from 12% (Toamasina) to 65% (Ambatondrazaka). The main risk factor associated with ESBL-E. coli carriage was the rainy season (OR = 2.9, 95% CI 1.3–5.6, p = 0.009). Whole genome sequencing was performed on 168 isolates from 144 participants. blaCTX–M–15 was the most frequent ESBL gene (86%). One isolate was resistant to carbapenems and carried the blaNDM–5 gene. Most isolates belonged to commensalism associated phylogenetic groups A, B1, and C (90%) and marginally to extra-intestinal virulence associated phylogenetic groups B2, D and F (10%). Multi locus sequence typing showed 67 different sequence types gathered in 17 clonal complexes (STc), the most frequent being STc10/phylogroup A (35%), followed distantly by the emerging STc155/phylogroup B1 (7%), STc38/phylogroup D (4%) and STc131/phylogroup B2 (3%). While a wide diversity of clones has been observed, SNP analysis revealed several genetically close isolates (n = 34/168) which suggests human-to-human transmissions. IncY plasmids were found with an unusual prevalence (23%), all carrying a blaCTX–M–15. Most of them (85%) showed substantial homology (≥85%) suggesting a dissemination of IncY ESBL plasmids in Madagascar. This large-scale study reveals a high prevalence of ESBL-E. coli among pregnant women in four cities in Madagascar associated with warmth and rainfall. It shows the great diversity of E. coli disseminating throughout the country but also transmission of specific clones and spread of plasmids. This highlights the urgent need of public-health interventions to control antibiotic resistance in the country.

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Genomic Diversity and Virulence Profiles of Historical Escherichia coli O157 Strains Isolated from Clinical and Environmental Sources

Applied and Environmental Microbiology ◽

10.1128/aem.02616-14 ◽

2014 ◽

Vol 81 (2) ◽

pp. 569-577 ◽

Cited By ~ 8

Author(s):

Lydia V. Rump ◽

Narjol Gonzalez-Escalona ◽

Wenting Ju ◽

Fei Wang ◽

Guojie Cao ◽

...

Keyword(s):

Escherichia Coli ◽

The United States ◽

Genomic Diversity ◽

Severe Illness ◽

Pathogenic Potential ◽

Content Type ◽

E Coli ◽

Virulence Markers ◽

Virulence Potential ◽

Food Borne

ABSTRACTEscherichia coliO157:H7 is, to date, the majorE. coliserotype causing food-borne human disease worldwide. Strains of O157 with other H antigens also have been recovered. We analyzed a collection of historic O157 strains (n= 400) isolated in the late 1980s to early 1990s in the United States. Strains were predominantly serotype O157:H7 (55%), and various O157:non-H7 (41%) serotypes were not previously reported regarding their pathogenic potential. Although lacking Shiga toxin (stx) andeaegenes, serotypes O157:H1, O157:H2, O157:H11, O157:H42, and O157:H43 carried several virulence factors (iha,terD, andhlyA) also found in virulent serotypeE. coliO157:H7. Pulsed-field gel electrophoresis (PFGE) showed the O157 serogroup was diverse, with strains with the same H type clustering together closely. Among non-H7 isolates, serotype O157:H43 was highly prevalent (65%) and carried important enterohemorrhagicE. coli(EHEC) virulence markers (iha,terD,hlyA, andespP). Isolates from two particular H types, H2 and H11, among the most commonly found non-O157 EHEC serotypes (O26:H11, O111:H11, O103:H2/H11, and O45:H2), unexpectedly clustered more closely with O157:H7 than other H types and carried several virulence genes. This suggests an early divergence of the O157 serogroup to clades with different pathogenic potentials. The appearance of important EHEC virulence markers in closely related H types suggests their virulence potential and suggests further monitoring of those serotypes not implicated in severe illness thus far.

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Prevalence and Genomic Characterization of Escherichia coli O157:H7 in Cow-Calf Herds throughout California

Applied and Environmental Microbiology ◽

10.1128/aem.00734-17 ◽

2017 ◽

Vol 83 (16) ◽

Cited By ~ 6

Author(s):

Jay N. Worley ◽

Kristopher A. Flores ◽

Xun Yang ◽

Jennifer A. Chase ◽

Guojie Cao ◽

...

Keyword(s):

Escherichia Coli ◽

Large Scale ◽

Fresh Produce ◽

Bacterial Pathogen ◽

Virulence Gene ◽

The United States ◽

Pathogenic Potential ◽

Content Type ◽

E Coli ◽

Cow Calf

ABSTRACT Escherichia coli serotype O157:H7 is a zoonotic food- and waterborne bacterial pathogen that causes a high hospitalization rate and can cause life-threatening complications. Increasingly, E. coli O157:H7 infections appear to originate from fresh produce. Ruminants, such as cattle, are a prominent reservoir of E. coli O157:H7 in the United States. California is one of the most agriculturally productive regions in the world for fresh produce, beef, and milk. The close proximity of fresh produce and cattle presents food safety challenges on a uniquely large scale. We performed a survey of E. coli O157:H7 on 20 farms in California to observe the regional diversity and prevalence of E. coli O157:H7. Isolates were obtained from enrichment cultures of cow feces. Some farms were sampled on two dates. Genomes from isolates were sequenced to determine their relatedness and pathogenic potential. E. coli O157:H7 was isolated from approximately half of the farms. The point prevalence of E. coli O157:H7 on farms was highly variable, ranging from zero to nearly 90%. Within farms, generally one or a few lineages were found, even when the rate of isolation was high. On farms with high isolation rates, a single clonal lineage accounted for most of the isolates. Farms that were visited months after the first visit might have had the same lineages of E. coli O157:H7. Strains of E. coli O157:H7 may be persistent for months on farms. IMPORTANCE This survey of 20 cow-calf operations from different regions of California provides an in depth look at resident Escherichia coli O157:H7 populations at the molecular level. E. coli O157:H7 is found to have a highly variable prevalence, and with whole-genome sequencing, high prevalences in herds were found to be due to a single lineage shed from multiple cows. Few repeat lineages were found between farms in this area; therefore, we predict that E. coli O157:H7 has significant diversity in this area beyond what is detected in this survey. All isolates from this study were found to have pathogenic potential based on the presence of key virulence gene sequences. This represents a novel insight into pathogen diversity within a single subtype and will inform future attempts to survey regional pathogen populations.

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LPS core type diversity in the Escherichia coli species and associations with phylogeny and virulence gene repertoire.

10.1101/2021.03.22.436425 ◽

2021 ◽

Author(s):

Sebastien Olivier Leclercq ◽

Maxime Branger ◽

David GE Smith ◽

Pierre GERMON

Keyword(s):

Escherichia Coli ◽

Lipid A ◽

Virulence Gene ◽

Uneven Distribution ◽

Gene Repertoire ◽

Phylogenetic Groups ◽

E Coli ◽

Wide Range ◽

External Part ◽

K 12

Escherichia coli is a very versatile species for which diversity has been explored from various perspectives highlighting, for example, phylogenetic groupings, pathovars as well as a wide range of O serotypes. The highly variable O-antigen, the most external part of the lipopolysaccharide component of the outer membrane of E. coli, is linked to the innermost lipid A through the core region of LPS of which 5 different structures, denominated K-12, R1, R2, R3 and R4, have been characterized so far. The aim of the present study was to analyze the prevalence of these LPS core types in the E. coli species and explore their distribution in the different E. coli phylogenetic groups and in relationship with the virulence gene repertoire. Results indicated an uneven distribution of core types between the different phylogroups, with phylogroup A strains being the most diverse in terms of LPS core types while phylogroups B1, D and E strains were dominated by the R3 type and phylogroups B2 and C strains being dominated by the R1 type. Strains carrying the LEE virulence operon were mostly of the R3 type whatever the phylogroup while, within phylogroup B2, strains carrying a K-12 core all belonged to the complex STc131, one of the major clone of extra-intestinal pathogenic E. coli(ExPEC) strains. The origin of this uneven distribution is discussed but remains to be explained, as well as the consequences of carrying a specific core type on the physiology of the bacteria.

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Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources

Applied and Environmental Microbiology ◽

10.1128/aem.00275-07 ◽

2007 ◽

Vol 73 (18) ◽

pp. 5703-5710 ◽

Cited By ~ 75

Author(s):

Satoshi Ishii ◽

Katriya P. Meyer ◽

Michael J. Sadowsky

Keyword(s):

Escherichia Coli ◽

Human Health ◽

Animal Species ◽

Virulence Gene ◽

Health Hazards ◽

Dna Fingerprint ◽

Phylogenetic Groups ◽

E Coli ◽

Animal Hosts ◽

Potential Human Health

ABSTRACT Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx 1c and/or stx 2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.

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Escherichia coli Specific Virulence-Gene Markers Analysis for Quality Control of Ovine Cheese in Slovakia

Microorganisms ◽

10.3390/microorganisms9091808 ◽

2021 ◽

Vol 9 (9) ◽

pp. 1808

Author(s):

Dobroslava Bujňáková ◽

Lívia Karahutová ◽

Vladimír Kmeť

Keyword(s):

Escherichia Coli ◽

Virulence Genes ◽

Disease Outbreaks ◽

Virulence Gene ◽

Phylogenetic Groups ◽

Gene Markers ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Ovine Cheese ◽

Phylogenetic Grouping

Shiga toxin-producing and extra-intestinal pathogenic Escherichia coli (E. coli) have the potential to spread through faecal waste, resulting in contamination of food and causing foodborne disease outbreaks. With the aim of characterizing unpasteurized ovine cheese in Slovakia, a total of 92 E. coli strains were examined for eleven representative virulence genes typical for (extra-)intestinal pathogenic E. coli and phylogenetic grouping. Phylogenetic groups B1 (36%) and A (32%) were the most dominant, followed by groups C (14%) and D (13%), while the lowest incidence was recorded for F (4%), and E (1%), and 43 (47%) samples carried at least one virulent gene, i.e., potential pathogens. Isolates present in groups E, F and D showed higher presence of virulence genes (100%, 75%, and 67%), versus 55%, 39%, and 28% in commensal B1, C, and A, respectively. Occurrence of papC and fyuA (both 24%) was highest, followed by tsh, iss, stx2, cnf1, kpsII, cvaC, stx1, iutA and eaeA. Nine E. coli strains (almost 10% of all tested and around 21% of our virulence-gene-associated isolates) harboured stx1, stx2 or eae. Ovine cheeses in Slovakia are highly contaminated with E. coli including potentially pathogenic strains capable of causing intestinal and/or extra-intestinal diseases, and thus may pose a threat to public health while unpasteurized.

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Clonal Relatedness, Phylotyping and Antimicrobial Susceptibility of Extended- spectrum-beta-lactamase Producing Uropathogenic Escherichia coli Isolates from Outpatients and Inpatients

Infectious Disorders - Drug Targets ◽

10.2174/1871526519666190715152118 ◽

2020 ◽

Vol 20 (5) ◽

pp. 659-666

Author(s):

Negar Azimzadeh ◽

Abdollah Derakhshandeh ◽

Mohammad Motamedifar ◽

Zahra Naziri

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Gel Electrophoresis ◽

Genomic Diversity ◽

Pulse Field ◽

Dominant Group ◽

Phylogenetic Groups ◽

Extended Spectrum Beta Lactamase ◽

E Coli ◽

Hospital Units

Objectives: Antibiotic resistance, phylogenetic groups and Pulsed-Field Gel Electrophoresis (PFGE) patterns were evaluated in urinary tract infection (UTI) Escherichia coli (E. coli) isolates from outpatients and inpatients. Methods: In this study, antibiotic resistance to E. coli isolated from non-hospitalized and hospitalized patients (153 outpatients and 147 inpatients ) was evaluated in Shiraz County, Iran. Phylogenetic groups and Pulse Field Gel Electrophoresis (PFGE) patterns of 143 ESBLs-producing E. coli were also assessed. Results: The prevalence of ESBL-producing E. coli was shown to be 46.4% and 49% in the outpatient and inpatient UTI E. coli isolates, respectively. Most ESBL-producers were detected on patients hospitalized in clinical surgery units (66.7%) and intensive care units (62.5%). Phylogenetic group D was the dominant group in both the outpatient and inpatient isolates (67.6% and 61.1%, respectively) and also in internal, clinical surgery and ICU units. PFGE results showed more relatedness (>80% similarity) among inpatient isolates. PFGE analysis of 49 ESBL-producing inpatient E.coli in hospital units revealed 17 different pulsotypes, consisting of 11 clones and 6 single patterns. There were no clonal patterns in outpatient isolates, and similarity among the outpatient isolates and also between inpatient and outpatient isolates was less than 80% (75% and 66%, respectively). Conclusions: The results showed extreme genomic diversity among the ESBL-producing E. coli isolates in terms of the community and multiclonal dissemination of ESBL-producing E. coli isolated from hospital units.

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Lineages and Virulence Gene Content among Extended-Spectrum β-Lactamase–Producing Escherichia coli Strains of Food Origin in Tunisia

Journal of Food Protection ◽

10.4315/0362-028x.jfp-12-251 ◽

2013 ◽

Vol 76 (2) ◽

pp. 323-327 ◽

Cited By ~ 18

Author(s):

AHLEM JOUINI ◽

KARIM BEN SLAMA ◽

NAOUEL KLIBI ◽

RYM BEN SALLEM ◽

VANESA ESTEPA ◽

...

Keyword(s):

Escherichia Coli ◽

Virulence Genes ◽

Virulence Gene ◽

Food Samples ◽

Phylogenetic Groups ◽

E Coli ◽

Extended Spectrum ◽

Sequence Types ◽

M 14 ◽

High Diversity

Nineteen extended-spectrum β-lactamase (ESBL)–positive Escherichia coli strains recovered from food samples in Tunisia were characterized by multilocus sequence typing and phylogenetic typing, and the virulence gene and plasmid content were also determined. These strains presented unrelated pulsed-field gel electrophoresis patterns and contained genes coding for the following ESBLs (the number of strains is in parentheses): CTX-M-1 (15), CTX-M-14 (2), CTX-M-8 (1), and SHV-5 (1). Twelve different sequence types (STs) were identified among the 19 ESBL-positive strains, which included two new STs (ST2022 in 2 blaCTX-M-14–containing strains and ST1970 in 2 blaCTX-M-1–containing strains). ST155 and ST602 were detected in four and three blaCTX-M-1–containing strains, respectively, and ST405 was detected in one blaCTX-M-8–producing strain. All ESBL-positive strains were ascribed to the phylogenetic groups A and B1. Most of the blaCTX-M-1–containing strains harbored an IncI1 plasmid, except for the four blaCTX-M-1–positive strains of beef origin and ST155, which harbored an IncN plasmid. The two blaCTX-M-14–containing strains contained an IncI1 plasmid. The virulence gene fimA was detected in all strains. Most strains also carried the aer gene, and six strains carried the eae gene. All strains were negative for the virulence genes sxt, papG-III, papC, hly, cnf1, and bfp. We conclude that ESBL-producing E. coli strains of food origin in Tunisia show high diversity and that plasmids harboring ESBL genes could be implicated in the dissemination of this resistance phenotype.

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Antimicrobial Resistance Glides in the Sky—Free-Living Birds as a Reservoir of Resistant Escherichia coli With Zoonotic Potential

Frontiers in Microbiology ◽

10.3389/fmicb.2021.656223 ◽

2021 ◽

Vol 12 ◽

Author(s):

Magdalena Skarżyńska ◽

Magdalena Zaja̧c ◽

Arkadiusz Bomba ◽

Łukasz Bocian ◽

Wojciech Kozdruń ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Animal Health ◽

Resistance Mechanisms ◽

Natural Habitats ◽

Free Living ◽

E Coli ◽

Virulence Potential ◽

Clonal Spread

Antimicrobial resistance (AMR) is one of the most important global health concerns; therefore, the identification of AMR reservoirs and vectors is essential. Attention should be paid to the recognition of potential hazards associated with wildlife as this field still seems to be incompletely explored. In this context, the role of free-living birds as AMR carriers is noteworthy. Therefore, we applied methods used in AMR monitoring, supplemented by colistin resistance screening, to investigate the AMR status of Escherichia coli from free-living birds coming from natural habitats and rescue centers. Whole-genome sequencing (WGS) of strains enabled to determine resistance mechanisms and investigate their epidemiological relationships and virulence potential. As far as we know, this study is one of the few that applied WGS of that number (n = 71) of strains coming from a wild avian reservoir. The primary concerns arising from our study relate to resistance and its determinants toward antimicrobial classes of the highest priority for the treatment of critical infections in people, e.g., cephalosporins, quinolones, polymyxins, and aminoglycosides, as well as fosfomycin. Among the numerous determinants, blaCTX–M–15, blaCMY–2, blaSHV–12, blaTEM–1B, qnrS1, qnrB19, mcr-1, fosA7, aac(3)-IIa, ant(3”)-Ia, and aph(6)-Id and chromosomal gyrA, parC, and parE mutations were identified. Fifty-two sequence types (STs) noted among 71 E. coli included the global lineages ST131, ST10, and ST224 as well as the three novel STs 11104, 11105, and 11194. Numerous virulence factors were noted with the prevailing terC, gad, ompT, iss, traT, lpfA, and sitA. Single E. coli was Shiga toxin-producing. Our study shows that the clonal spread of E. coli lineages of public and animal health relevance is a serious avian-associated hazard.

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Immunogold labeling of CMP-KDO synthetase produced by recombinant E. Coli cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100128900 ◽

1987 ◽

Vol 45 ◽

pp. 924-925

Author(s):

F. A. Durum ◽

R. G. Goldman ◽

T. J. Bolling ◽

M. F. Miller

Keyword(s):

Inclusion Bodies ◽

Large Scale ◽

Recombinant Dna ◽

Test System ◽

Cell Protein ◽

Gram Negative Bacteria ◽

Cytoplasmic Inclusions ◽

Isolation And Purification ◽

E Coli ◽

Low Concentrations

CMP-KDO synthetase (CKS) is an enzyme which plays a key role in the synthesis of LPS, an outer membrane component unique to gram negative bacteria. CKS activates KDO to CMP-KDO for incorporation into LPS. The enzyme is normally present in low concentrations (0.02% of total cell protein) which makes it difficult to perform large scale isolation and purification. Recently, the gene for CKS from E. coli was cloned and various recombinant DNA constructs overproducing CKS several thousandfold (unpublished data) were derived. Interestingly, no cytoplasmic inclusions of overproduced CKS were observed by EM (Fig. 1) which is in contrast to other reports of large proteinaceous inclusion bodies in various overproducing recombinant strains. The present immunocytochemical study was undertaken to localize CKS in these cells.Immune labeling conditions were first optimized using a previously described cell-free test system. Briefly, this involves soaking small blocks of polymerized bovine serum albumin in purified CKS antigen and subjecting them to various fixation, embedding and immunochemical conditions.

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