TWiM #284: Flies, Squid and Pigs

1. Paper Abstracts

1.1. Snippet paper; discussion starts at 5:11 minutes

“The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig feces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, ‘and TEM-1’ was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2 km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man.” (Behrens et al. 2023, no changes)

1.2. Main paper; discussion starts at 25:29 minutes

“Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host’s natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes.” (McAnulty et al. 2023, no changes)

2. Vision and Change Core Concepts and 2024 ASM Fundamental Statements

3.  Potential Learning Objectives for the Podcast

¹Papers: Snippet (S) or Main (M)
²Learning Objectives: Lower Order or Higher Order (H)

4. Techniques Described (with Time Stamps)

Here is a link to a bio-dictionary that has many, but not all definitions if you need a definition: Explore Biology Bio-Dictionary

4.1. Snippet Paper

• Whole Genome Sequencing (11:28–11:59; 15:57–16:06): Whole genome sequencing is a next generation sequencing (NGS) technique to sequence a whole genome, compare it to a reference genome, and identify changes between them.  Here it was used to identify base pair sequences related to antibiotic-resistant characteristics within bacteria acquired from the pigs and flies and in microbial source tracking, i.e., determining the microbe’s origin.
• Multi-locus Sequence Type (MLST) Analysis (14:55–x15:35): Multi-locus sequence typing is a method of using the sequence of a set of housekeeping genes to determine relatedness of bacterial species.  Here it was utilized in comparing relationships of bacterial sequences isolated from pig feces and the flies.
• Antibiotic Susceptibility Testing (16:07–16:30; 17:06–17:25): This is an assay to determine the degree to which a bacterium is sensitive to an antibiotic. Here, it was used to show the dispersal of pig-associated antibiotic resistant bacteria.
• Fluorescent/Dye Tagging (19:06–19:22): Fluorescent molecules can be used to track a number of things.  Here, flies were painted with fluorescent dyes, and their migratory patterns tracked to measure transmission range.
• Pulsed-Field Gel Electrophoresis (19:40–20:06): This is an electrophoresis technique to separate large DNA fragments.  Here, it was used to discussed that Salmonella sp. and Escherichia coli have indistinguishable DNA macro-restriction patterns [pulsotypes].

4.2. Main Paper

• Model Organism (28:09–28:15; 44:28–44:40; 47:10–47:15): A model organism is a specific organism with a set of features that is extensively studied; the findings from studying this organism are used to hypothesize the characteristics or relationships of other organisms.
• Gnotobiosis (32:50–33:16): This is a method for rearing/culturing organisms under laboratory conditions that remove all other associated organisms (parasites, microbiota, etc.).  Here is was used so that squid microflora could be manipulated.
• Microbiota Manipulation  (35:40–37:15): These methods involved using different sources of microbes to populate the squid microbiota.  Here, various sand collections were placed under different conditions to examine microbial population and its subsequent effects on the squid. Some sand batches were autoclaved, and others were left out to dry in the sun to decrease microbial populations.
• Reporter/Indicator Bacteria (37:30–38:43): These are bacteria that are biomarkers.  Here, bacteria that produce blue colonies were used to indicate and measure the effectiveness of “spiking” wild wet sand.
• Beta Diversity (40:48–43:12): This is a measure of the diversity of a region based on species composition of the area.  Low diversity indicates that communities in the region are more similar and high diversity indicates that communities are less alike.  Here, it was visually represented with Bray Curtis beta diversity graphs, the closeness of points to each other indicates less biodiversity.
• 16S rDNA Genomic Analysis (43:18–44:45): Metagenomic sequencing of 16S rDNA is a “high-level, low-resolution” sequencing technique to identify present bacterial taxa present in a mixed sample.  It is unable to differentiate at more precise (genera, species) levels.

5. Connections to General Microbiology Processes/Concepts (with Time Stamps)

5.1. Snippet Paper

5.2. Main Paper

• Mutualism (28:16–29:12; 30:36–31:50): Vibrio fischeri at high density can show bioluminescence. E. scolopes has developed a light organ colonized by the bacteria that aids in protection from predators.
• Quorum Sensing (29:50–30:26): Vibrio fischeri secrete small molecules to sense neighboring cells. When the bacterial population is high, gene expression is induced to produce Lux genes responsible for bioluminescence.
• Microbial Ecology (33:22–34:34; 38:00–39:03): Naturally occurring bacterial populations in the sand found in the environment of the squid are responsible for supplying the squid with the bacteria needed for effective reproduction.
• Consortial Microbiomes (35:01–37:29): Bacterial communities differ in the ANG of the squid depending on the sand available where they were collected or raised. Changes to the bacterial community affected the effectiveness of the sand and the reproductive outcome within the squid.
• Adaptive Immunity (46:25–47:02): The immune system requires exposure to different antigens in order to create specialized defenses (e.g. antibodies) in response; this complex process of exposure and development is paralleled to the growth of the squid’s reproductive organ

The following are two figure reading exercises, one from the snippet paper (Figure 3) and one from the main paper (Figure 5AB).

7.1. First Figure Reading Exercise

Experimental Background (Behrens et al., Figure 3)

Antimicrobial uses in agriculture extend beyond treating infections because antibiotic exposure increases animal growth, making the time to market shorter.  Since antimicrobial resistance evolves by exposure and resistance to current antimicrobials is a major concern because it is responsible for many deaths worldwide, Behrens et al. (2023) investigated the prevalence of agriculturally-based antibiotic resistant bacteria at animal farms, and also possible dispersal by insect vectors.  To do this, they focused on pig farms where piglets are treated with colistin, ciprofloxacin, and cefotaxime to fatten them, but also are treated with other antibiotics to treat infections.  They collected feces from piglets in twelve herds when they arrived at each farm and then every week or two weeks for a total of eight weeks.  They next quantified the number of resistant bacteria in the feces.  They identified 336 antibiotic resistant Escherichia coli isolates associated with eleven different drug classes (Beta Lactams, Aminoglycosides, Diaminopyrimidine, Fosfomycin, Macrolides and Lincosamides, Fluoroquinolone, Peptide, Sulfonamide, Eflamycin, Phenicol, and Nucleoside).  They also found that viable antibiotic-resistant bacteria could be found in barn compartments and the open stable.  So, their next experiment focused on dispersal from the pigs to the structural buildings, closed stables.  They quantified the proportion of living enterobacteria that were resistant to colistin, ciprofloxacin, and cefotaxime in farm dust (left side of panel) and in houseflies (right side of panel) found on the farm.

7.1.2. Questions

1. Living bacteria resistant to each antibiotic is quantified by its proportion and represented in a box plot.  Match the box plot feature with its description. (1 = mean; 2 = median; 3 = outlier; 4 = interquartile range; 5 = individual measures; 6= 1.5 x the interquartile range)
   1. ______ Horizontal line in box
   2. ______ Symbols, such as circles
   3. ______ Box
   4. ______ Whiskers
2. Which sample has the largest variability in sample values? What is your evidence?
   1. dust, ciprofloxacin; the median is highest
   2. dust, colistin; the range is smallest
   3. fly, ciprofloxacin; there is an outlier dot
   4. fly, cefotaxime; the interquartile box is largest
3. Based on these data, is dust from the closed stable a possible source of antibiotic resistance transmission? What is your evidence?
   1. yes; at least 10% of the bacteria are resistant to each antibiotic
   2. yes; there is significantly more resistance to cefotaxime
   3. no; there is no difference for the three antibiotic resistance levels
   4. no; there is no difference in antibiotic resistance compared to flies
4. Based on these data, are houseflies a possible source of antibiotic resistance transmission? What is your evidence?
   1. no; there is significantly less resistance to the antibiotic cefotaxime
   2. no; there is no difference in antibiotic resistance compared to dust
   3. yes; there is statistically significant levels of resistance to all antibiotics
   4. yes; at least for ciprofloxacin and colistin, but maybe not for cefotaxime
5. Given that houseflies were found 2 kilometers from the farm, which antibiotic resistances are the most cause for concern based on these data? [pick all that apply]
   1. ciprofloxacin
   2. colistin
   3. cefotaxime