This work is funded by UNM IMSD (NIH-NIGMS) grant #GM060201, the UNM el centro de la RAZA IME scholarship, and the UNM Department of Biology Alvin R. and Caroline G. Grove Summer Research Scholarship, Kenneth Ingham Consulting, and John Craig.

 

Searching for Novel Antibiotics in Caves1Department of Biology, University of New Mexico, MSC03 2020, Albuquerque, NM 87131-0001

Elizabeth T. Montano1, Jesse H. Alvarez1, Diana E. Northup1, Margaret C. Werner-Washburne1, and Eric C. Toolson1

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Fig. 2 Cave isolates that inhibited common pathogens were tested against one another. Inhibition may indicate the production of dissimilar compounds whereas no inhibition may indicate similar compounds. No data are isolates that rendered unclear results and will be re-tested.

Research Questions

SignificanceNew antibiotics are needed as resistance develops and persists. There are a limited number of antibiotics and bacteria develop resistance to these over time. There is a compelling need to identify novel antibiotics. New antibiotics have been discovered in bacterial screens but an unexploited potential source of novel antibiotics exists in caves. In the deeper parts of the caves the bacteria have been undisturbed for perhaps millions of years and thus potentially represent separate evolutionary trajectories and might be expected to produce different types of antibiotics.

Methods

4 Carbonate Caves in SE New Mexico

Results: All-Against-All

Results: Reciprocal Inhibition

Conclusions

Acknowledgements

References1. Arias CA, Murray BE (2009) Antibiotic-resistant bugs in the 21st century - a clinical superchallenge. N Engl J Med. doi: 10.5495/wjcid.v1.i1.11 2. Davies J (2006) Are antibiotics naturally antibiotics? J Ind Microbiol Biotechnol. doi: 10.1007/s10295-006-0112-5 3. Groth I, Vetterman RB, Scheuetzte SP, Saiz-Jimenez C (1999) Actinomycetes in karstic caves of northern Spain Altamira, and Tito Bustillo. J Microbiol Methods. doi: 10.1016/S0167-7012(99)00016-0 4. Lertcanawanichakul M, Sawangnop S (2008) A comparison of two methods used for measuring the antagonistic activity of Bacillus species. Walailak J Sci & Tech 5:161–171 5. Peláez F (2006) The historical delivery of antibiotics from microbial natural products-can history repeat? Biochem Pharmacol. doi: 10.1016/j.bcp. 2005.10.010

Future Research

1. Do cave bacteria, which can grow in isolation over millions of years and, thus, may represent independent evolutionary trajectories, produce novel chemicals involved in communication, including antibiotics?

2. Can we use ecological characteristics such as depth, nutrients, humidity, etc., to predict where high diversity and/or production of novel antibiotics is likely to occur?

CavesNumber Tested

Number Positive

Antimicrobial Activity

Depth Carbon NitrogenDegree of Dark Zone

Backcountry Cave 247 28 11% -1.5-6m 10.35% 0.258% *

Spider Cave 202 23 11% -1-24m 7.81% 0.032% ***

Carlsbad Cavern: Left Hand Tunnel

(CLHT)40 5 12% -228m 4.98% 0.035% *****

Lechuguilla Cave 203 52 26%

-150-

300m12.4% 0.050% *****

Inhibition No Inhibition No Data

Reciprocal Inhibition No Reciprocal Inhibition

Fig. 3 Reciprocal inhibition (yellow cells) is a pair of cave isolates that were mutually inhibitory. This was not the case for all isolates that were sensitive to one another and those are shown here as the blue cells. The double arrow lines along the bottom of the figure indicate reciprocal pairs of isolates from the same cave where depth increases along both axes.

Lechuguilla SpiderCLHTBackcountry

1. A few strains, which inhibit their own growth, appear to use general mechanisms and inhibit a wide range of isolates 2. Isolates that show some overlap in inhibition patterns, do not show complete overlap, suggesting they are producing distinct antimicrobials3. Two separate caves, Spider and Lechuguilla, had higher than expected reciprocal inhibitions (suggesting they produce distinct antimicrobials) and production appears to increase with increasing depth and may represent independent evolutionary trajectories.

Future studies include testing this set of isolates against a suite of common antibiotics. The isolates that display sensitivity will be targeted for whole genome sequencing followed by mining for novel gene clusters that may confer new pathways for novel antimicrobial production.

All-against-all inhibition tests were carried out to identify strains that showed reciprocal inhibition and to look for similarities in inhibition patterns

The most significant reciprocal inhibition occurs between caves

Lechuguilla SpiderCLHTBackcountryTable 1. Antimicrobial activity among cave isolates and abiotic factors (depth and average nutrient percentages) that could influence antimicrobial activity. Degree of dark zone ranges from * to ***** (* is very little )

Results: Pathogen Inhibition

Cave isolates were tested against common pathogens to identify strains producing antimicrobial compounds and to look for patterns of inhibition

Fig1. The most frequently inhibited pathogens in all four caves were Gram-negative;Klebsiella pneumoniae ATCC 13883 , Proteus vulgaris ATCC 13315 , and Shigella flexneri ATCC 9199. Cultured isolates from Lechuguilla Cave were highly effective against Proteus vulgaris. Gram-negative pathogens were inhibited almost three times more often as G+

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Data Analysis Model• Isolates 4 & 7 are mutual inhibitors (reciprocal pair)• Isolates 4 & 5 are a non-reciprocal pair• Isolate 8 inhibited itself and many others and is likely using a general mechanism

Lech-Lech Back-Back

LHT-LHT

Spid-Spid

Lech-Back

Lech-LHT

Lech-Spid

Back-LHT

Back-Spid

LHT-Spid

Observed 50 10 0 19 52 17 72 11 20 1Expected 58.42 16.94 0.54 11.43 62.91 11.23 51.68 6.05 27.83 4.97

Cell Chi-square 1.21 2.84 0.54 5.02 1.89 2.96 7.99 4.05 2.20 3.17

Total Chi-square 31.88df 9p 0.0002

Fig, 4 There are noticeably more Reciprocal Inhibitions than expected when Lechuguilla isolates are tested against Spider isolates (Lech-Spid), and fewer than expected when Lechuguilla isolates are tested against Back isolates (Lech-Back).

A Chi-square test was applied to determine whether the reciprocal inhibitions were significantly non-randomly distributed