Search

Abstract Background Cronobacter sakazakii is a foodborne pathogen that causes septicemia, meningitis, and necrotizing enterocolitis in neonates and infants. The current research details the full genome sequences of two extremely persistent C. sakazakii strains (H322 and GK1025B) isolated from powdered infant formula (PIF) manufacturing settings. In addition, the genetic attributes associated with five plasmids, pH322_1, pH322_2, pGK1025B_1, pGK1025B_2, and pGK1025B_3 are described. Materials and Methods Using PacBio single-molecule real-time (SMRT®) sequencing technology, whole genome sequence (WGS) assemblies of C. sakazakii H322 [Sequence type (ST)83, clonal complex [CC] 83) and GK1025B (ST64, CC64) were generated. Plasmids, also sequenced, were aligned with phylogenetically related episomes to determine, and identify conserved and missing genomic regions. Results A truncated ~ 13 Kbp type 6 secretion system (T6SS) gene cluster harbored on virulence plasmids pH322_2 and pGK1025B_2, and a second large deletion (~ 6 Kbp) on pH322_2, which included genes for a tyrosine-type recombinase/integrase, a hypothetical protein, and a phospholipase D was identified. Within the T6SS of pH322_2 and pGK1025B_2, an arsenic resistance operon was identified which is in common with that of plasmids pSP291_1 and pESA3. In addition, PHASTER analysis identified an intact 96.9 Kbp Salmonella SSU5 prophage gene cluster in pH322_1 and pGK1025B_1 and showed that these two plasmids were phylogenetically related to C. sakazakii plasmids: pCS1, pCsa767a, pCsaC757b, pCsaC105731a. Plasmid pGK1025B_3 was identified as a novel conjugative Cronobacter plasmid. Furthermore, WGS analysis identified a ~ 16.4 Kbp type 4 secretion system gene cluster harbored on pGK1025B_3, which contained a phospholipase D gene, a key virulence factor in several host–pathogen diseases. Conclusion These data provide high resolution information on C. sakazakii genomes and emphasizes the need for furthering surveillance studies to link genotype to phenotype of strains from previous investigations. These results provide baseline data necessary for future in-depth investigations of C. sakazakii that colonize PIF manufacturing facility settings and genomic analyses of these two C. sakazakii strains and five associated plasmids will contribute to a better understanding of this pathogen's survival and persistence within various “built environments” like PIF manufacturing facilities.

Antimicrobial resistance is a global health challenge. Few new antibiotics have been developed for use over the years, and preserving the efficacy of existing compounds is an important step to protect public health. This paper describes a study that examines the effects of exogenously induced oxidative stress on K. pneumoniae

Cronobacter sakazakii continues to be isolated from ready-to-eat fresh and frozen produce, flours, dairy powders, cereals, nuts, and spices, in addition to the conventional sources of powdered infant formulae (PIF) and PIF production environments. To understand the sequence diversity, phylogenetic relationship, and virulence of C. sakazakii originating from plant-origin foods, comparative molecular and genomic analyses, and zebrafish infection (ZI) studies were applied to 88 strains. Whole genome sequences of the strains were generated for detailed bioinformatic analysis. PCR analysis showed that all strains possessed a pESA3-like virulence plasmid similar to reference C. sakazakii clinical strain BAA-894. Core genome analysis confirmed a shared genomic backbone with other C. sakazakii strains from food, clinical and environmental strains. Emerging nucleotide diversity in these plant-origin strains was highlighted using single nucleotide polymorphic alleles in 2000 core genes. DNA hybridization analyses using a pan-genomic microarray showed that these strains clustered according to sequence types (STs) identified by multi-locus sequence typing (MLST). PHASTER analysis identified 185 intact prophage gene clusters encompassing 22 different prophages, including three intact Cronobacter prophages: ENT47670, ENT39118, and phiES15. AMRFinderPlus analysis identified the CSA family class C β-lactamase gene in all strains and a plasmid-borne mcr-9.1 gene was identified in three strains. ZI studies showed that some plant-origin C. sakazakii display virulence comparable to clinical strains. Finding virulent plant-origin C. sakazakii possessing significant genomic features of clinically relevant STs suggests that these foods can serve as potential transmission vehicles and supports widening the scope of continued surveillance for this important foodborne pathogen.

Cronobacter species are opportunistic pathogens capable of causing life-threatening infections in humans, with serious complications arising in neonates, infants, immuno-compromised individuals, and elderly adults. The genus is comprised of seven species: Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter muytjensii, Cronobacter dublinensis, Cronobacter universalis, and Cronobacter condimenti. Despite a multiplicity of genomic data for the genus, little is known about likely transmission vectors. Using DNA microarray analysis, in parallel with whole genome sequencing, and targeted PCR analyses, the total gene content of two C. malonaticus, three C. turicensis, and 14 C. sakazaki isolated from various filth flies was assessed. Phylogenetic relatedness among these and other strains obtained during surveillance and outbreak investigations were comparatively assessed. Specifically, microarray analysis (MA) demonstrated its utility to cluster strains according to species-specific and sequence type (ST) phylogenetic relatedness, and that the fly strains clustered among strains obtained from clinical, food and environmental sources from United States, Europe, and Southeast Asia. This combinatorial approach was useful in data mining for virulence factor genes, and phage genes and gene clusters. In addition, results of plasmidotyping were in agreement with the species identity for each strain as determined by species-specific PCR assays, MA, and whole genome sequencing. Microarray and BLAST analyses of Cronobacter fly sequence datasets were corroborative and showed that the presence and absence of virulence factors followed species and ST evolutionary lines even though such genes were orthologous. Additionally, zebrafish infectivity studies showed that these pathotypes were as virulent to zebrafish embryos as other clinical strains. In summary, these findings support a striking phylogeny amongst fly, clinical, and surveillance strains isolated during 2010–2015, suggesting that flies are capable vectors for transmission of virulent Cronobacter spp.; they continue to circulate among United States and European populations, environments, and that this “pattern of circulation” has continued over decades.

Abstract Cronobacter sakazakii is a Gram-negative opportunistic pathogen that causes life- threatening infantile infections, such as meningitis, septicemia, and necrotizing enterocolitis, as well as pneumonia, septicemia, and urinary tract and wound infections in adults. Here, we report 26 draft genome sequences of C. sakazakii, which were obtained from dried spices from the USA, the Middle East, China, and the Republic of Korea. The average genome size of the C. sakazakii genomes was 4393 kb, with an average of 4055 protein coding genes, and an average genome G + C content of 56.9%. The genomes contained genes related to carbohydrate transport and metabolism, amino acid transport and metabolism, and cell wall/membrane biogenesis. In addition, we identified genes encoding proteins involved in osmotic responses such as DnaJ, Aquaproin Z, ProQ, and TreF, as well as virulence-related and heat shock-related proteins. Interestingly, a metabolic island comprised of a variably-sized xylose utilization operon was found within the spice-associated C. sakazakii genomes, which supports the hypothesis that plants may serve as transmission vectors or alternative hosts for Cronobacter species. The presence of the genes identified in this study can support the remarkable phenotypic traits of C. sakazakii such as the organism’s capabilities of adaptation and survival in response to adverse growth environmental conditions (e.g. osmotic and desiccative stresses). Accordingly, the genome analyses provided insights into many aspects of physiology and evolutionary history of this important foodborne pathogen.

Abstract Background Malonate utilization, an important differential trait, well recognized as being possessed by six of the seven Cronobacter species is thought to be largely absent in Cronobacter sakazakii (Csak). The current study provides experimental evidence that confirms the presence of a malonate utilization operon in 24 strains of sequence type (ST) 64, obtained from Europe, Middle East, China, and USA; it offers explanations regarding the genomic diversity and phylogenetic relatedness among these strains, and that of other C. sakazakii strains. Results In this study, the presence of a malonate utilization operon in these strains was initially identified by DNA microarray analysis (MA) out of a pool of 347 strains obtained from various surveillance studies involving clinical, spices, milk powder sources and powdered infant formula production facilities in Ireland and Germany, and dried dairy powder manufacturing facilities in the USA. All ST64 C. sakazakii strains tested could utilize malonate. Zebrafish embryo infection studies showed that C. sakazakii ST64 strains are as virulent as other Cronobacter species. Parallel whole genome sequencing (WGS) and MA showed that the strains phylogenetically grouped as a separate clade among the Csak species cluster. Additionally, these strains possessed the Csak O:2 serotype. The nine-gene, ~ 7.7 kbp malonate utilization operon was located in these strains between two conserved flanking genes, gyrB and katG. Plasmidotyping results showed that these strains possessed the virulence plasmid pESA3, but in contrast to the USA ST64 Csak strains, ST64 Csak strains isolated from sources in Europe and the Middle East, did not possess the type six secretion system effector vgrG gene. Conclusions Until this investigation, the presence of malonate-positive Csak strains, which are associated with foods and clinical cases, was under appreciated. If this trait was used solely to identify Cronobacter strains, many strains would likely be misidentified. Parallel WGS and MA were useful in characterizing the total genome content of these Csak O:2, ST64, malonate-positive strains and further provides an understanding of their phylogenetic relatedness among other virulent C. sakazakii strains.

ABSTRACT Listeria monocytogenes is frequently found in foods and processing facilities, where it can persist, creating concerns for the food industry. Its ability to survive under a wide range of environmental conditions enhances the potential for cross-contamination of the final food products, leading to possible outbreaks of listeriosis. In this study, whole-genome sequencing (WGS) was applied as a tool to characterize and track 100 L. monocytogenes isolates collected from three food processing environments. These WGS data from environmental and food isolates were analyzed to (i) assess the genomic diversity of L. monocytogenes, (ii) identify possible source(s) of contamination, cross-contamination routes, and persistence, (iii) detect absence/presence of antimicrobial resistance-encoding genes, (iv) assess virulence genotypes, and (v) explore in vivo pathogenicity of selected L. monocytogenes isolates carrying different virulence genotypes. The predominant L. monocytogenes sublineages (SLs) identified were SL101 (21%), SL9 (17%), SL121 (12%), and SL5 (12%). Benzalkonium chloride (BC) tolerance-encoding genes were found in 62% of these isolates, a value that increased to 73% among putative persistent subgroups. The most prevalent gene was emrC followed by bcrABC, qacH-Tn6188, and qacC. The L. monocytogenes major virulence factor inlA was truncated in 31% of the isolates, and only one environmental isolate (L. monocytogenes CFS086) harbored all major virulence factors, including Listeria pathogenicity island 4 (LIPI-4), which has been shown to confer hypervirulence. A zebrafish embryo infection model showed a low (3%) embryo survival rate for all putatively hypervirulent L. monocytogenes isolates assayed. Higher embryo survival rates were observed following infection with unknown virulence potential (20%) and putatively hypovirulent (53 to 83%) L. monocytogenes isolates showing predicted pathogenic phenotypes inferred from virulence genotypes. IMPORTANCE This study extends current understanding of the genetic diversity among L. monocytogenes from various food products and food processing environments. Application of WGS-based strategies facilitated tracking of this pathogen of importance to human health along the production chain while providing insights into the pathogenic potential for some of the L. monocytogenes isolates recovered. These analyses enabled the grouping of selected isolates into three putative virulence categories according to their genotypes along with informing selection for phenotypic assessment of their pathogenicity using the zebrafish embryo infection model. It has also facilitated the identification of those isolates with genes conferring tolerance to commercially used biocides. Findings from this study highlight the potential for the application of WGS as a proactive tool to support food safety controls as applied to L. monocytogenes.

Cronobacter species are considered an opportunistic group of foodborne pathogenic bacteria capable of causing both intestinal and systemic human disease. This review describes common virulence themes shared among the seven Cronobacter species and describes multiple exoproteins secreted by Cronobacter, many of which are bacterial toxins that may play a role in human disease. The review will particularly concentrate on the virulence factors secreted by C. sakazakii, C. malonaticus, and C. turicensis, which are the primary human pathogens of interest. It has been discovered that various species-specific virulence factors adversely affect a wide range of eukaryotic cell processes including protein synthesis, cell division, and ion secretion. Many of these factors are toxins which have been shown to also modulate the host immune response. These factors are encoded on a variety of mobile genetic elements such as plasmids and transposons; this genomic plasticity implies ongoing re-assortment of virulence factor genes which has complicated our efforts to categorize Cronobacter into sharply defined genomic pathotypes.

Cronobacter (C.) sakazakii is an opportunistic pathogen and has been associated with serious infections with high mortality rates predominantly in pre-term, low-birth weight and/or immune compromised neonates and infants. Infections have been epidemiologically linked to consumption of intrinsically and extrinsically contaminated lots of reconstituted powdered infant formula (PIF), thus contamination of such products is a challenging task for the PIF producing industry. We present the draft genome of C. sakazakii H322, a highly persistent sequence type (ST) 83, clonal complex (CC) 65, serotype O:7 strain obtained from a batch of non-released contaminated PIF product. The presence of this strain in the production environment was traced back more than 4 years. Whole genome sequencing (WGS) of this strain together with four more ST83 strains (PIF production environment-associated) confirmed a high degree of sequence homology among four of the five strains. Phylogenetic analysis using microarray (MA) and WGS data showed that the ST83 strains were highly phylogenetically related and MA showed that between 5 and 38 genes differed from one another in these strains. All strains possessed the pESA3-like virulence plasmid and one strain possessed a pESA2-like plasmid. In addition, a pCS1-like plasmid was also found. In order to assess the potential in vivo pathogenicity of the ST83 strains, each strain was subjected to infection studies using the recently developed zebrafish embryo model. Our results showed a high (90–100%) zebrafish mortality rate for all of these strains, suggesting a high risk for infections and illness in neonates potentially exposed to PIF contaminated with ST83 C. sakazakii strains. In summary, virulent ST83, CC65, serotype CsakO:7 strains, though rarely found intrinsically in PIF, can persist within a PIF manufacturing facility for years and potentially pose significant quality assurance challenges to the PIF manufacturing industry.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swimbladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signalling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Bacteria belonging to the genus Cronobacter have been recognized as causative agents of life-threatening systemic infections primarily in premature, low-birth weight and immune-compromised neonates. Apparently not all Cronobacter species are linked to infantile infections and it has been proposed that virulence varies among strains. Whole genome comparisons and in silico analysis have proven to be powerful tools in elucidating potential virulence determinants, the presence/absence of which may explain the differential virulence behaviour of strains. However, validation of these factors has in the past been hampered by the availability of a suitable neonatal animal model. In the present study we have used zebrafish embryos to model Cronobacter infections in vivo using wild type and genetically engineered strains. Our experiments confirmed the role of the RepF1B-like plasmids as "virulence plasmids" in Cronobacter and underpinned the importantce of two putative virulence factors-cpa and zpx-in in vivo pathogenesis. We propose that by using this model in vivo infection studies are now possible on a large scale level which will boost the understanding on the virulence strategies employed by these pathogens.

Bacillus thuringiensis var. israelensis (Bti) and Lysinibacillus sphaericus (Lsph) are extensively used in mosquito control programs. These biocides are the active ingredients of a commercial larvicide. Quantitative data on the fate of both Bti and Lsph applied together for the control of mosquitoes in urban drainage structures such as catch basins are lacking. We evaluated the dynamics and persistence of Bti and Lsph spores released through their concomitant application in urban catch basins in southern Switzerland. Detection and quantification of spores over time in water and sludge samples from catch basins were carried out using quantitative real-time PCR targeting both cry4A and cry4B toxin genes for Bti and the binA gene for Lsph. After treatment, Bti and Lsph spores attained concentrations of 3.76 (± 0.08) and 4.13 (± 0.09) log ml(-1) in water, then decreased progressively over time, reaching baseline values. For both Bti and Lsph, spore levels in the order of 10(5) g(-1) were observed in the bottom sludge two days after the treatment and remained constant for the whole test period (275 days). Indigenous Lsph strains were isolated from previously untreated catch basins. A selection of those was genotyped using pulsed field gel electrophoresis of SmaI-digested chromosomal DNA, revealing that a subset of isolates were members of the clonal population of strain 2362. No safety issues related to the use of this biopesticide in the environment have been observed during this study, because no significant increase in the number of spores was seen during the long observation period. The isolation of native Lysinibacillus sphaericus strains belonging to the same clonal population as strain 2362 from catch basins never treated with Lsph-based products indicates that the use of a combination of Bti and Lsph for the control of mosquitoes does not introduce non-indigenous microorganisms in this area.

Author(s): Marc Strous [1]; Eric Pelletier [2]; Sophie Mangenot [2]; Thomas Rattei [3]; Angelika Lehner [4, 5]; Michael W. Taylor [4]; Matthias Horn [4]; Holger Daims [4]; Delphine Bartol-Mavel [2]; [...]

A 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes is developed and applied for the parallel detection of all bacteria of the order 'Rhodocyclales' at different hierarchical or identical phylogenetic levels. The microarray results demonstrated the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.