Abstract Cronobacter spp. are opportunistic foodborne pathogens associated with infections in neonates and infants, particularly those that are premature or immunocompromised. Outbreaks of Cronobacter infections in infants have been epidemiologically linked to the consumption of contaminated powdered infant formulae. Since the designation of Enterobacter sakazakii (now Cronobacter spp.) as a unique species in 1980, a number of methods have been proposed for its detection and identification. The aim of the present study was to evaluate and compare different tools used for the identification of presumptive Cronobacter isolates. The bioMérieux API 20E biochemical bacterial identification kit (Omnimed (Pty), Randburg, Gauteng, South Africa) was shown not to be a reliable identification tool for the Cronobacter strains examined in the current study, since it gave false-positive results. The API 50CHE biochemical kit was shown to be more reliable giving similar results to the polymerase chain reaction (PCR) detection and DNA sequence data. The primer pair Esakf/Esakr proved to be the most reliable PCR identification tool.Additional differentiating traits and antibiotic patterns were demonstrated for Cronobacter species in the current study.

The purpose of this research was to explore the potential application of lytic phages to control Cronobacter spp. in infant formula. More than two hundred and fifty phages were isolated from various environmental samples against different strains of Cronobacter spp. Selected phages were characterized by morphology, host range, and cross infectivity. The genomes of five novel Cronobacter phages [vB_CsaM_GAP31 (GAP31), vB_CsaM_GAP32 (GAP32), vB_CsaP_GAP52 (GAP52), vB_CsaM_GAP161 (GAP161), vB_CsaP_GAP227 (GAP227)] were sequenced. Phage GAP32 possess the second largest phage genome sequenced to date, and it is proposed that GAP32 belongs to a new genus of “Gap32likeviruses”. Phages GAP52 and GAP227 are the first C. sakazakii podoviruses whose genomes have been sequenced. None of the sequenced genomes showed homology to virulent or lysogenic genes. In addition, in vivo administration of phage GAP161 in the hemolymph of Galleria mellonella larvae showed no negative effects on the wellbeing of the larvae and could effectively prevent Cronobacter infection in the larvae. A cocktail of five phages was highly effective for biocontrol of three Cronobacter sakazakii strains present as a mixed culture in both broth media and contaminated reconstituted infant formula. This phage cocktail could be potentially used to control C. sakazakii during preparation of infant formula but would first have to be clinically evaluated in mammalian models. / NSERC & DFO

Made available in DSpace on 2014-08-26T17:15:00Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 60.pdf: 1087420 bytes, checksum: d43c3b6da28a66b2ee5f2ae5c11fc7ae (MD5) Previous issue date: 2010 / Cronobacter spp. (Enterobacter sakazakii) são patógenos emergentes associados a surtos de meningite, enterocolite necrosante e septicemia em neonatos em diversos países. A taxa de mortalidade relatada varia de 40 a 80% e os sobreviventes apresentam seqüelas neurológicas severas. O desenvolvimento da infecção está associado ao consumo de fórmulas infantis desidratadas. As metodologias atualmente disponíveis para o isolamento de Cronobacter spp. apresentam discrepâncias e a identificação bioquímica não é esclarecedora. Assim, torna-se necessário que metodologias confiáveis para o isolamento e identificação desses micro-organismos estejam disponíveis. Outro ponto ainda não esclarecido são os mecanismos de virulência utilizados por esses patógenos para o desenvolvimento de infecções em humanos. Os objetivos desse estudo foram o desenvolvimento de um método rápido e eficiente para a identificação de Cronobacter spp., a avaliação do perfil de resistência a antimicrobianos e a avaliação da presença de alguns fatores de virulência. Foram utilizadas inicialmente cepas de referência de Cronobacter spp e 37 isolados previamente identificados como pertencentes ao gênero. O novo protocolo foi considerado 100% sensível e específico quando comparado aos demais métodos utilizados, indicando sua utlidade. O estudo de fatores de virulência revelou que Cronobacter spp. tem desenvolvido resistência aos β-lactâmicos e cefalosporinas, é capaz de formar cápsula, biofilme, e apresentar atividade de protease e atividade hemolítica. Nesse estudo relata-se pela primeira vez a presença de hemaglutininas associadas a pili tipo 3, enquanto algumas cepas apresentaram hemaglutininas associadas a pili tipo 1. Também foi demonstrada a atividade citotóxica em células Vero e a capacidade de invasão dessas células por Cronobacter spp. Os resultados desse estudo sugerem que todas as espécies do gênero Cronobacter apresentam potencial patogênico e que autoridades de saúde pública, produtores de alimentos e pessoas responsáveis pelo cuidado de indivíduos que fazem parte dos grupos de risco devem dedicar especial atenção ao controle de ambientes e à qualidade microbiológica de produtos destinados a esses grupos de modo a minimizar o risco de infecção por esses micro-organismos. / Cronobacter spp. (Enterobacter sakazakii) is an emerging foodborne pathogen associated with outbreaks of meningitis, necrotizing enterocolitis and sepsis in neonates in different countries. The reported mortality rate ranges from 40 to 80% and the survivors present severe neurological sequela. Development of the infection is associated with the consumption of powdered infant formula. The available methodologies employed for the isolation of Cronobacter spp. lack the necessary specificity and the biochemical identification remains non-conclusive. For these reasons, it is necessary to develop reliable methodologies for the isolation and identification of these microorganisms. Another issue still to be clarified refers to the virulence mechanisms associated with human infections. The objectives of this study were to develop a rapid and efficient method to identify Cronobacter, to evaluate the antimicrobial resistance of the strains, and to evaluate the presence of some virulence factors. Initially, reference strains of Cronobacter, and 37 isolates that had been previously identified as members of the genus were used. The proposed protocol showed 100% sensitivity and specificity when compared to the other methods used, indicating its usefulness. Studies on virulence factors revealed that Cronobacter spp. has developed resistance to β-lactams and cephalosporins, is able to form capsule, biofilm, and presents protease and hemolytic activities. This study reports, for the first time in literature, the presence of hemagglutinins associated with type 3 pili and it was also shown that some strains presented previously described hemagglutinins associated with type 1 pili. Cytotoxic activity in Vero cells and the invasiveness of these cells by Cronobacter spp were also demonstrated. The results of this study suggest that all species of the genus Cronobacter have pathogenic potential and public health authorities, food producers and caregivers should pay careful attention to issues associated with environment control and with the microbial quality of the products associated with the risk groups in order to minimize the risk of infection with these microorganisms.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter. / AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak. Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle virulensie. Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee afsonderlike groepe gedeel met 'n 93% vertrouens vlak. Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr. muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr. malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met malonaatbenutting-analises. 'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme (BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon. DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig. Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf spesies van Cronobacter kan verseker.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-21T09:27:02Z (GMT). No. of bitstreams: 1 Araujo_FranciscaDianadaSilva_D.pdf: 5387746 bytes, checksum: ae8110c020cf470d09fb0e22f670508f (MD5) Previous issue date: 2012 / Resumo: Aspectos distintos da ecologia química de bactéria, fungo e abelha foram investigados e abordados em três capítulos. O primeiro capítulo descreve o estudo das moléculas sinalizadoras envolvidas no processo de quorum-sensing da bactéria Enterobacter sakazakii (Cronobacter spp.), que resultou na identificação de três acil-homosserina lactonas (acil-HSLs): (S)-(-)-N-heptanoil-HSL, (S)-(-)-N-dodecanoil-HSL e (S)-(-)-N-tetradecanoil-HSL. Estes semioquímicos foram degradados por enzimas de Bacillus cereus. No segundo capítulo o estudo do fungo Epicoccum nigrum possibilitou o isolamento de meleína, 4,5-dimetil-resorcinol, flavipina e de um novo metabólito, denominado epicolactona. O monitoramento destas substâncias em três mutantes de E. nigrum apontou para a produção de 5-hidroxi-meleína, ausente no fungo selvagem, indicando que a rota biossintética de policetídeos em E. nigrum foi alterada, ativando a ação de uma mono-oxigenase responsável pela oxidação da meleína. No terceiro capítulo, foram investigados feromônios produzidos por diferentes castas da abelha social Tetragonisca angustula. Bioensaios com as rainhas indicaram que lipídios cuticulares são possíveis responsáveis pela indução da cópula nos machos. Operárias fundadoras e guardas foram diferenciadas quimicamente por compostos presentes em seus extratos cefálicos e abdominais. Extratos cefálicos de machos em diferentes ciclos de vida mostraram-se semelhantes / Abstract: Different aspects of bacteria, fungus and bee chemical ecology were investigated and discussed in three chapters. First chapter describes the study of signaling molecules involved in the Enterobacter sakazakii (Cronobacter spp.) quorum sensing process that resulted in the identification of three acyl-homoserine lactones (acyl-HSLs): (S)-(-)-N-heptanoyl-HSL, (S)-(-)-N-dodecanoyl-HSL and (S)-(-)-N-tetradecanoyl-HSL. These semiochemicals were modified by Bacillus cereus enzymes. Second chapter describes the study of Epicoccum nigrum (fungus) revealing the isolation of mellein, 4,5-dimethylresorcinol, flavipin and of a novel metabolite, named epicolactone. Monitoring these compounds in three E. nigrum mutants pointed to the production of 5-hydroxymellein, absent in wild fungus strain, indicating that biosynthetic route of polyketides in E. nigrum has been modified, activating a monooxygenase responsible for mellein oxidation. Third chapter describes pheromones produced by different castes of Tetragonisca angustula social bee. Bioassays with queens indicated that cuticular lipids might be responsible for copulation induction in males. Cephalic and abdominal extracts of founded and guard workers were chemically different, while cephalic extracts of males at different life cycles were similar / Doutorado / Quimica Organica / Doutora em Ciências

Les produits alimentaires secs sont courants dans l’industrie agroalimentaire. Cependant, leur innocuité n’est pas toujours assurée et de nombreux cas de toxi-infections alimentaires collectives à travers le monde sont annuellement recensés. Deux bactéries pathogènes sont particulièrement impliquées, l’une correspond à grand nombre de cas, Salmonella enterica, et l’autre est reconnue pour sa capacité à résister aux perturbations environnementales, Cronobacter spp.. Une compréhension plus poussée de l’impact du séchage et traitement thermique à l’état sec peut permettre une optimisation des procédés de de décontamination des produits alimentaires secs et contribuer à réduire le nombre de toxiinfections alimentaires. Dans un premier chapitre, une attention particulière a été portée au procédé de séchage de microorganismes pathogènes qui peut, s’il est conduit de manière rapide, réduire considérablement la cultivabilité microbienne. De cette façon, le séchage peut être considéré comme une étape supplémentaire de décontamination à condition d’en optimiser les conditions d’application. Puis dans un deuxième chapitre, l’impact de la réhydratation, rarement traitée jusqu’à présent, a été étudié afin de proposer des conditions permettant de maximiser l’inactivation bactérienne. Cette étude remet partiellement en cause les dénombrements de bactéries pathogènes dans les aliments qui peuvent être, dans quelque cas, sous estimées du fait de l’utilisation de réhydratation rapide. Dans un troisième chapitre, l’application de différents traitements thermiques sur du lait en poudre, destiné à une population dite sensible, a permis de comprendre le rôle couplé de l’activité de l’eau et de la température sur l’efficacité du traitement thermique à l’état sec mais également de proposer un modèle d’inactivation thermique des pathogènes. Il a été notamment possible de définir un paramètre représentatif de l’effet de l’activité de l’eau (yaw). Les mécanismes de mort cellulaire pour les deux bactéries ont été ensuite étudiés, que ce soit après un séchage ou après un traitement thermique. Il apparait clairement que le séchage induit majoritairement une perméabilisation de la membrane tandis que le traitement thermique des cellules viables séchées dégrade majoritairement l’activité enzymatique en détruisant peu la membrane. Finalement, il a été montré que la virulence des deux pathogènes étudiés est augmentée par le séchage mais que le traitement thermique à l’état sec n’a pas d’impact sur cette virulence. Les connaissances apportées par cette étude peuvent être utiles d’un point de vue industriel, afin d’optimiser au mieux les conditions d’application des différents procédées pour une inactivation bactérienne maximale. / Dried food products are common in food industry. Nevertheless, their safety is not well insured, involving numerous outbreaks every years around the world. Particularly, two pathogenic bacteria are of interest, one of them due to its number of cases, Salmonella enterica, and the other one due to its ability to survive environmental perturbations, Cronobacter spp.. A deeper comprehension of drying and heat treatment in dried state impact could lead to an optimization of drying and heating processes, insuring food safety. In the first instance, drying was considered as a supplementary decontamination step by optimizing its conditions of use. In a second phase, the rehydration impact was studied in order to found optimal conditions of pathogen inactivation. The study questions the rehydration currently puts in practice for food safety analysis. In a third phase, several heat treatments on a dried food product, intended to susceptible population, permitted to understand the specific role of water activity on the efficiency of heat treatment in dried state, and also propose a modelling for bacterial heat inactivation. In addition, it was possible to define two new parameters which represent the effect of temperature (zT) and the effect of water activity (yaw). Fourth, cellular death mechanisms for both studied bacteria, during the drying or during the heat treatment, were considered. It was clear that drying involved mainly membrane damages whereas heat treatment involved mainly enzymatic damages. Finally, in a fifth part, it was shown that virulence properties of both studied bacteria were affected by drying, increasing invasion capacity, but not by heat treatment in dried state. To finish, the knowledges brought by this study are useful for industrial point of view, in the case of drying and heat process optimization and also by proposing biological indicators to valid process efficiency in situ.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

Cronobacter sakazakii is a gram-negative opportunistic pathogen known to survive in dry environments and food matrices, such as infant formula. This foodborne bacterium can cause fatal human infections of the blood, central nervous system, and gastrointestinal tract; it is also problematic in wounds and urinary tract infections. Preterm infants and immunocompromised individuals are in higher risk categories related to necrotizing enterocolitis, neonatal sepsis, and meningitis due to this organism. Therefore, there is a need for increased understanding of how this bacterium is able to persist in thermally treated low-moisture products that do not support growth. The objective of this research is to identify genes and mechanisms in C. sakazakii that contribute to its resistance to desiccation and survival in low-moisture food matrices, including powdered infant formula. C. sakazakii sequence type 4 (ST4) is of particular interest as it is often the cause of neonatal infections originating from contaminated feedings of powder infant formula. The method chosen to explore these genetic patterns is massively parallel transposon insertion sequencing (Tn-seq). The E. coli strain MFDpir was used to facilitate transposon insertional mutagenesis to create a library of mutated C. sakazakii. Three different C. sakazakii ST4 isolates of different origins (clinical, environmental, and infant formula-derived) were selected for this study. Once transposon mutagenesis occurred with the aid of E. coli MFDpir, the three mutant libraries were subjected to desiccation stress in a closed system equilibrated to 11.3% relative humidity. The surviving mutant genomes were analyzed with Tn-seq. The sequencing data revealed that, while transposition events did occur successfully within the genomes of each of the selected C. sakazakii isolates, these events were not dense enough to draw biological conclusions nor statistical inferences concerning which genes contribute to this organism’s uncanny desiccation tolerance. However, we concluded that the Tn-seq method is a promising tool with this organism of interest, despite incomplete results in this first round of experimentation.