Complete Genome Sequence of UV-Resistant \u3ci\u3eCampylobacter jejuni\u3c/i\u3e RM3194, Including an 81.08-Kilobase Plasmid

Annually, Campylobacter spp. are responsible for the greatest number of foodborne gastrointestinal bacterial infections in the developed world (1, 2). However, Campylobacter spp. are nutritionally fastidious organisms requiring a microaerobic environment for survival (3). It remains uncertain by what mechanisms Campylobacter survives within multiple hostile environments in sufficient numbers to cause such significant amounts of human disease. Campylobacter jejuni strain RM3194 was isolated from a clinical sample in 1994 from a human enteritis case at the Red Cross War Memorial Children’s Hospital in Cape Town, South Africa, and was supplied to our laboratory by Robert Mandrell (ARS, Albany, CA) (4, 5). In our research, RM3194 demonstrated an increased resistance to both UV (254 nm) and blue light (405 nm) (6). This resistance produced several-log-greater survival after challenge with UVlight compared to the survival of other C. jejuni strains in our collection

Complete Genome Sequence of UV-Resistant \u3ci\u3eCampylobacter jejuni\u3c/i\u3e RM3194, Including an 81.08-Kilobase Plasmid

Annually, Campylobacter spp. are responsible for the greatest number of foodborne gastrointestinal bacterial infections in the developed world (1, 2). However, Campylobacter spp. are nutritionally fastidious organisms requiring a microaerobic environment for survival (3). It remains uncertain by what mechanisms Campylobacter survives within multiple hostile environments in sufficient numbers to cause such significant amounts of human disease. Campylobacter jejuni strain RM3194 was isolated from a clinical sample in 1994 from a human enteritis case at the Red Cross War Memorial Children’s Hospital in Cape Town, South Africa, and was supplied to our laboratory by Robert Mandrell (ARS, Albany, CA) (4, 5). In our research, RM3194 demonstrated an increased resistance to both UV (254 nm) and blue light (405 nm) (6). This resistance produced several-log-greater survival after challenge with UVlight compared to the survival of other C. jejuni strains in our collection

Association of \u3ci\u3eEscherichia coli\u3c/i\u3e O157:H7 \u3ci\u3etir\u3c/i\u3e polymorphisms with human infection

Background: Emerging molecular, animal model and epidemiologic evidence suggests that Shigatoxigenic Escherichia coli O157:H7 (STEC O157) isolates vary in their capacity to cause human infection and disease. The translocated intimin receptor (tir) and intimin (eae) are virulence factors and bacterial receptor-ligand proteins responsible for tight STEC O157 adherence to intestinal epithelial cells. They represent logical genomic targets to investigate the role of sequence variation in STEC O157 pathogenesis and molecular epidemiology. The purposes of this study were (1) to identify tir and eae polymorphisms in diverse STEC O157 isolates derived from clinically ill humans and healthy cattle (the dominant zoonotic reservoir) and (2) to test any observed tir and eae polymorphisms for association with human (vs bovine) isolate source. Results: Five polymorphisms were identified in a 1,627-bp segment of tir. Alleles of two tir polymorphisms, tir 255 T\u3eA and repeat region 1-repeat unit 3 (RR1-RU3, presence or absence) had dissimilar distributions among human and bovine isolates. More than 99% of 108 human isolates possessed the tir 255 T\u3eA T allele and lacked RR1-RU3. In contrast, the tir 255 T\u3eA T allele and RR1-RU3 absence were found in 55% and 57%, respectively, of 77 bovine isolates. Both polymorphisms associated strongly with isolate source (p \u3c 0.0001), but not by pulsed field gel electrophoresis type or by stx1 and stx2 status (as determined by PCR). Two eae polymorphisms were identified in a 2,755-bp segment of 44 human and bovine isolates; 42 isolates had identical eae sequences. The eae polymorphisms did not associate with isolate source. Conclusion: Polymorphisms in tir but not eae predict the propensity of STEC O157 isolates to cause human clinical disease. The over-representation of the tir 255 T\u3eA T allele in human-derived isolates vs the tir 255 T\u3eA A allele suggests that these isolates have a higher propensity to cause disease. The high frequency of bovine isolates with the A allele suggests a possible bovine ecological niche for this STEC O157 subset

Locus of Heat Resistance (LHR) in Meat-Borne \u3ci\u3eEscherichia coli\u3c/i\u3e: Screening and Genetic Characterization

Microbial resistance to processing treatments poses a food safety concern, as treatment tolerant pathogens can emerge. Occasional foodborne outbreaks caused by pathogenic Escherichia coli have led to human and economic losses. Therefore, this study screened for the extreme heat resistance (XHR) phenotype as well as one known genetic marker, the locus of heat resistance (LHR), in 4,123 E. coli isolates from diverse meat animals at different processing stages. The prevalences of XHR and LHR among the meat-borne E. coli were found to be 10.3% and 11.4%, respectively, with 19% agreement between the two. Finished meat products showed the highest LHR prevalence (24.3%) compared to other processing stages (0 to 0.6%). None of the LHR1 E. coli in this study would be considered pathogens based on screening for virulence genes. Four high-quality genomes were generated by whole-genome sequencing of representative LHR+ isolates. Nine horizontally acquired LHRs were identified and characterized, four plasmid-borne and five chromosomal. Nine newly identified LHRs belong to ClpK1 LHR or ClpK2 LHR variants sharing 61 to 68% nucleotide sequence identity, while one LHR appears to be a hybrid. Our observations suggest positive correlation between the number of LHR regions present in isolates and the extent of heat resistance. The isolate exhibiting the highest degree of heat resistance possessed four LHRs belonging to three different variant groups. Maintenance of as many as four LHRs in a single genome emphasizes the benefits of the LHR in bacterial physiology and stress response. IMPORTANCE Currently, a “multiple-hurdle” approach based on a combination of different antimicrobial interventions, including heat, is being utilized during meat processing to control the burden of spoilage and pathogenic bacteria. Our recent study (M. Guragain, G. E. Smith, D. A. King, and J. M. Bosilevac, J Food Prot 83:1438–1443, 2020, https://doi.org/10.4315/JFP-20-103) suggests that U.S. beef cattle harbor Escherichia coli that possess the locus of heat resistance (LHR). LHR seemingly contributes to the global stress tolerance in bacteria and hence poses a food safety concern. Therefore, it is important to understand the distribution of the LHRs among meat-borne bacteria identified at different stages of different meat processing systems. Complete genome sequencing and comparative analysis of selected heat-resistant bacteria provide a clearer understanding of stress and heat resistance mechanisms. Further, sequencing data may offer a platform to gain further insights into the genetic background that provides optimal bacterial tolerance against heat and other processing treatments

Phylogenetic classification of Escherichia coli O157:H7 strains of human and bovine origin using a novel set of nucleotide polymorphisms

Novel SNPs from human and bovine O157:H7 E. coli isolates are mapped, revealing that the majority of human disease is caused by a bovine subset of this strain

Evolution of a zoonotic pathogen:investigating prophage diversity in enterohaemorrhagic Escherichia coli O157 by long-read sequencing

Enterohaemorrhagic Escherichia coli (EHEC) O157 is a zoonotic pathogen for which colonization of cattle and virulence in humans is associated with multiple horizontally acquired genes, the majority present in active or cryptic prophages. Our understanding of the evolution and phylogeny of EHEC O157 continues to develop primarily based on core genome analyses; however, such short-read sequences have limited value for the analysis of prophage content and its chromosomal location. In this study, we applied Single Molecule Real Time (SMRT) sequencing, using the Pacific Biosciences long-read sequencing platform, to isolates selected from the main sub-clusters of this clonal group. Prophage regions were extracted from these sequences and from published reference strains. Genome position and prophage diversity were analysed along with genetic content. Prophages could be assigned to clusters, with smaller prophages generally exhibiting less diversity and preferential loss of structural genes. Prophages encoding Shiga toxin (Stx) 2a and Stx1a were the most diverse, and more variable compared to prophages encoding Stx2c, further supporting the hypothesis that Stx2c-prophage integration was ancestral to acquisition of other Stx types. The concept that phage type (PT) 21/28 (Stx2a+, Stx2c+) strains evolved from PT32 (Stx2c+) was supported by analysis of strains with excised Stx-encoding prophages. Insertion sequence elements were over-represented in prophage sequences compared to the rest of the genome, showing integration in key genes such as stx and an excisionase, the latter potentially acting to capture the bacteriophage into the genome. Prophage profiling should allow more accurate prediction of the pathogenic potential of isolates

The RR Lyrae variables and Horizontal Branch of NGC6656 (M22)

The first calibrated broadband UBVI time-series photometry is presented for the RR Lyrae variable stars in NGC6656 (M22), with observations spanning a range of twenty-two years. We have also redetermined the variability types and periods for the RR Lyrae stars identified previously by photographic observations, revising the number of fundamental-mode RR Lyrae variables (RR0) to 10 and the number of first-overtone variables (RR1) to 16. The mean periods of the RR0 and RR1 variables are _{RR0}=0.66 \pm 0.02 d and _{RR1}=0.33 \pm 0.01 d, respectively, supporting an Oosterhoff II classification for the cluster. The number ratio of RR1- to all RR-type variables is N_1/N_{RR}=0.61, also consistent with an Oosterhoff II designation. Both the RR Lyrae stars' minimum light colors and the blue edge of the RR Lyrae instability strip suggest E(B-V)=0.36 \pm 0.02 mag toward M22. Regarding the HB morphology of M22, we find (B-R)/(B+V+R)=+0.97 \pm 0.1 and at least one "gap" located in an unusual part of the blue HB, in the middle of the so-called hot HB stars.Comment: accepted to A

Transduction by Phi Bb-1, a Bacteriophage of Borrelia Burgdorferi

We previously described a bacteriophage of the Lyme disease agent Borrelia burgdorferi designated phi BB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus Borrelia. To demonstrate the ability of phi BB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into B. burgdorferi CA-11.2A cells. The kan cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the kan cassette was packaged by phi BB-1 released from this B. burgdorferi strain. phi BB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of B. burgdorferi. This is the first direct evidence of a mechanism for lateral gene transfer in B. burgdorferi