Molecular Basis of Antibiotic Self-Resistance in a Bee Larvae Pathogen

Paenibacillus larvae, the causative agent of the devastating honey-bee disease American Foulbrood, produces the cationic polyketide-peptide hybrid paenilamicin that displays high antibacterial and antifungal activity. Its biosynthetic gene cluster contains a gene coding for the N-acetyltransferase PamZ. We show that PamZ acts as self-resistance factor in P. larvae by deactivation of paenilamicin. Using tandem MS, NMR spectroscopy and synthetic diastereomers, we identified the N-terminal amino group of the agmatinamic acid as the N-acetylation site. These findings highlight the pharmacophore region of paenilamicin, which we very recently identified as a new ribosome inhibitor. Here, we further elucidated the crystal structure of PamZ:acetyl-CoA complex at 1.34 Å resolution. An unusual tandem-domain architecture provides a well-defined substrate-binding groove decorated with negatively-charged residues to specifically attract the cationic paenilamicin. Our results will help to understand the mode of action of paenilamicin and its role in pathogenicity of P. larvae to fight American Foulbrood.

American foulbrood and its causative agent, Paenibacillus larvae, in New Zealand’s registered hives and apiaries

<p>Though the honey bee (Apis mellifera) is exposed to an extensive diversity of parasites and pathogens from multiple kingdoms, few are as devastating as American foulbrood. American foulbrood is a highly contagious bacterial disease, of which the causative agent (bacterium Paenibacillus larvae) infects honey bee brood through the ingestion of its spores, ultimately leading to the death of the infected larva and the collapse of the infected hive. Paenibacillus larvae’s genotypes (ERIC I-IV) exhibit differing ‘killing time’ of infected larvae, resulting in different larval and colony level virulence of the disease within hives. American foulbrood is found in New Zealand’s registered hives, and poses a threat to the country’s apiculture industry. The first objective of this thesis was to perform a genetic analysis on New Zealand’s P. larvae field strains using the well-established methodology of rep-PCR with MBO REP1 primers. A total of 172 bacteria isolates were gathered from registered hives from 2011 to 2014 and examined. The MBO REP1 primer identifies the ‘beta’ genetic subgroups of P. larvae. By identifying beta subgroups, the ERIC genotypes that are present in New Zealand can also be concluded. The genetic analysis of P. larvae using rep-PCR is a first for New Zealand, and appears to be a first for Australasia. The second objective of this thesis was to conduct a temporal and geographical statistical analysis on American foulbrood infection rate trends in New Zealand’s national and regional, divided into seven regions, registered hives and apiaries from 1994 to 2013. The genetic analysis of P. larvae detected three ‘beta’ genotypic subgroups: B, b, and Б. From these findings it was concluded that ERIC I and ERIC II are present in New Zealand. Previous to my findings, subgroup B and Б and ERIC II genotype had not been recorded outside of Europe. The statistical analysis reported that American foulbrood infection rates were significantly decreasing nationally. Results also reported that four of the seven regions’ infection rates were significantly decreasing, whilst three regions were significantly increasing. Conclusions on the subgroups and genotypes present in New Zealand gives the first insight to the virulence and occurrence of P. larvae strains. Additionally, the use of rep-PCR for the genetic analysis of P. larvae enables this thesis to contribute to the increasing knowledge on American foulbrood. By examining the temporal and geographic dynamics of American foulbrood, the results allow for the evaluation of current management strategies and the most recent understanding on the national and regional infection rates of the disease.</p>

American foulbrood and its causative agent, Paenibacillus larvae, in New Zealand’s registered hives and apiaries

<p>Though the honey bee (Apis mellifera) is exposed to an extensive diversity of parasites and pathogens from multiple kingdoms, few are as devastating as American foulbrood. American foulbrood is a highly contagious bacterial disease, of which the causative agent (bacterium Paenibacillus larvae) infects honey bee brood through the ingestion of its spores, ultimately leading to the death of the infected larva and the collapse of the infected hive. Paenibacillus larvae’s genotypes (ERIC I-IV) exhibit differing ‘killing time’ of infected larvae, resulting in different larval and colony level virulence of the disease within hives. American foulbrood is found in New Zealand’s registered hives, and poses a threat to the country’s apiculture industry. The first objective of this thesis was to perform a genetic analysis on New Zealand’s P. larvae field strains using the well-established methodology of rep-PCR with MBO REP1 primers. A total of 172 bacteria isolates were gathered from registered hives from 2011 to 2014 and examined. The MBO REP1 primer identifies the ‘beta’ genetic subgroups of P. larvae. By identifying beta subgroups, the ERIC genotypes that are present in New Zealand can also be concluded. The genetic analysis of P. larvae using rep-PCR is a first for New Zealand, and appears to be a first for Australasia. The second objective of this thesis was to conduct a temporal and geographical statistical analysis on American foulbrood infection rate trends in New Zealand’s national and regional, divided into seven regions, registered hives and apiaries from 1994 to 2013. The genetic analysis of P. larvae detected three ‘beta’ genotypic subgroups: B, b, and Б. From these findings it was concluded that ERIC I and ERIC II are present in New Zealand. Previous to my findings, subgroup B and Б and ERIC II genotype had not been recorded outside of Europe. The statistical analysis reported that American foulbrood infection rates were significantly decreasing nationally. Results also reported that four of the seven regions’ infection rates were significantly decreasing, whilst three regions were significantly increasing. Conclusions on the subgroups and genotypes present in New Zealand gives the first insight to the virulence and occurrence of P. larvae strains. Additionally, the use of rep-PCR for the genetic analysis of P. larvae enables this thesis to contribute to the increasing knowledge on American foulbrood. By examining the temporal and geographic dynamics of American foulbrood, the results allow for the evaluation of current management strategies and the most recent understanding on the national and regional infection rates of the disease.</p>

Anti-Virulence Strategy against the Honey Bee Pathogenic Bacterium Paenibacillus larvae via Small Molecule Inhibitors of the Bacterial Toxin Plx2A

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

American Foulbrood in the Czech Republic: ERIC II Genotype of Paenibacillus Larvae Is Prevalent

American foulbrood (AFB) is a dangerous disease of honeybees (Apis mellifera) caused by the spore-forming bacterium Paenibacillus larvae. According to the ERIC (enterobacterial repetitive intergenic consensus) classification, five genotypes are distinguished, i.e., I, II, III, IV, and V, which differ in their virulence and prevalence in colonies. In the Czech Republic, AFB prevalence is monitored by the State Veterinary Administration; however, the occurrence of specific P. larvae genotypes within the country remains unknown. In this study, our aim was to genotype field P. larvae strains collected in the Czech Republic according to the ERIC classification. In total, 102 field isolates from colonies with AFB clinical symptoms were collected from various locations in the Czech Republic, and the PCR genotypization was performed using ERIC primers. We confirmed the presence of both ERIC I and II genotypes, while ERIC III, IV, and V were not detected. The majority of samples (n = 82, 80.4%) were identified as ERIC II, while the ERIC I genotype was confirmed only in 20 samples (19.6%). In contrast to other European countries, the ERIC II genotype is predominant in Czech honeybee colonies. The ERIC I genotype was mostly detected in border regions close to Poland, Slovakia, and Austria.

Recording the Presence of Peanibacillus larvae larvae Colonies on MYPGP Substrates Using a Multi-Sensor Array Based on Solid-State Gas Sensors

American foulbrood is a dangerous disease of bee broods found worldwide, caused by the Paenibacillus larvae larvae L. bacterium. In an experiment, the possibility of detecting colonies of this bacterium on MYPGP substrates (which contains yeast extract, Mueller-Hinton broth, glucose, K2HPO4, sodium pyruvate, and agar) was tested using a prototype of a multi-sensor recorder of the MCA-8 sensor signal with a matrix of six semiconductors: TGS 823, TGS 826, TGS 832, TGS 2600, TGS 2602, and TGS 2603 from Figaro. Two twin prototypes of the MCA-8 measurement device, M1 and M2, were used in the study. Each prototype was attached to two laboratory test chambers: a wooden one and a polystyrene one. For the experiment, the strain used was P. l. larvae ATCC 9545, ERIC I. On MYPGP medium, often used for laboratory diagnosis of American foulbrood, this bacterium produces small, transparent, smooth, and shiny colonies. Gas samples from over culture media of one- and two-day-old foulbrood P. l. larvae (with no colonies visible to the naked eye) and from over culture media older than 2 days (with visible bacterial colonies) were examined. In addition, the air from empty chambers was tested. The measurement time was 20 min, including a 10-min testing exposure phase and a 10-min sensor regeneration phase. The results were analyzed in two variants: without baseline correction and with baseline correction. We tested 14 classifiers and found that a prototype of a multi-sensor recorder of the MCA-8 sensor signal was capable of detecting colonies of P. l. larvae on MYPGP substrate with a 97% efficiency and could distinguish between MYPGP substrates with 1–2 days of culture, and substrates with older cultures. The efficacy of copies of the prototypes M1 and M2 was shown to differ slightly. The weighted method with Canberra metrics (Canberra.811) and kNN with Canberra and Manhattan metrics (Canberra. 1nn and manhattan.1nn) proved to be the most effective classifiers.

Isolation and Characterization of Phages Active against Paenibacillus larvae Causing American Foulbrood in Honeybees in Poland

The aim of this study was the isolation and characterization, including the phage effect on honeybees in laboratory conditions, of phages active against Paenibacillus larvae, the causative agent of American Foulbrood—a highly infective and easily spreading disease occurring in honeybee larva, and subsequently the development of a preparation to prevent and treat this dangerous disease. From the tested material (over 2500 samples) 35 Paenibacillus spp. strains were obtained and used to search for phages. Five phages specific to Paenibacillus were isolated and characterized (ultrastructure, morphology, biological properties, storage stability, and genome sequence). The characteristics were performed to obtain knowledge of their lytic potential and compose the final phage cocktail with high antibacterial potential and intended use of future field application. Preliminary safety studies have also been carried out on healthy bees, which suggest that the phage preparation administered is harmless.