Industrial insect mass rearing aims to produce quality insects under safe sanitary conditions which can be compromised by pathogens and abiotic stressors. Therefore, knowledge on pathogen persistence, virulence and means of detection is of importance. This study focuses on the opportunistic pathogen Serratia marcescens (Sm) as a possible candidate to reveal sanitary issues in Tenebrio molitor (Tm) breeding. A screening test was performed to assess the impact of abiotic stressors (starvation, density and sieving) in presence and absence of Sm. Two Sm detection methods were conducted, and the kinetics of Sm persistence were investigated. Our results show that (i) the presence of Sm had a low but significant effect on Tm mortality, (ii) a short temporary starvation period had a negative impact on larval growth, (iii) the detection of Sm by q-PCR was sensitive but less convenient than a specific Sm growth media, (iv) the kinetics of persistence showed that Sm declined but survived for nine days in the feed and in the feces for three weeks. Both the relatively low virulence and the persistence in the environment suggest that Sm could be used as an indicator for the sanitary status of mealworm production.

Bacillus cereus is a Gram-positive opportunistic pathogen closely related to the entomopathogen, Bacillus thuringiensis, both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of B. cereus genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free Galleria mellonella model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of G. mellonella encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in B. cereus gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific in situ gene expression analysis of extracellular bacteria in a whole animal.

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP–CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD₅₀) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD₅₀ is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

(2017). Is there a transgenerational inheritance of host resistance against pathogens? Lessons from the Galleria mellonella-Bacillus thuringiensis interaction model. Virulence: Vol. 8, No. 8, pp. 1471-1474.

Lactobacillus sakeiis a non-pathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment,L. sakeidoes not need iron or heme for growth, while possessing a heme-dependent catalase. Iron incorporation intoL. sakeifrom myoglobin and hemoglobin was formerly shown by microscopy and theL. sakeigenome reveals a complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (lsa1836-1840) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might refer to a complete machinery of an Energy Coupling Factor (ECF) transport system. We quantifiedin vitrothe intracellular heme in wild-type (WT) and in our Δlsa1836-1840deletion mutant using an intracellular heme sensor and ICP-Mass spectrometry for quantifying incorporated⁵⁷Fe heme. We showed that in the WTL. sakei, heme accumulation occurs fast and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored byin transcomplementation. Our results establish the main role of theL. sakeiLsa1836-1840 ECF-like system in heme uptake. This research outcome shed new light on other possible functions of ECF-like systems.Importance: Lactobacillus sakeiis a non-pathogenic bacterial species exhibiting high fitness in heme rich environments such as meat products, although it does not need iron nor heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered as virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for non-pathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacteriumL. sakei.In this study, we demonstrate that one of them, is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF, therefore our study reveals a new class of heme transport system.