Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation

Nosho, Kazuki; Yasuhara, Koji; Ikehata, Yuto; Mii, Tomohiro; Ishige, Taichiro; Yajima, Seishi; Hidaka, Masaaki; Ogawa, Tetsuhiro; Hara, Masaki

doi:10.1099/mic.0.000673

Cited by 13 publications

(9 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Secondly, viable but nonculturable (VBNC) L. pneumophila on agar plates may be able to reproduce in Legiolert liquid medium. Nosho et al (2018) showed the presence of Escherichia coli that cannot form colonies on agar plates but grows only in liquid medium. Thus, there may be L. pneumophila that grow well in the liquid medium but not on agar plates.…”

mentioning

confidence: 99%

Evaluation of Legiolert for Quantification of <i>Legionella pneumophila</i> from Bath Water Samples

Inoue¹,

Baba²,

Tayama³

2020

Biocontrol Sci.

View full text Add to dashboard Cite

Monitoring of Legionella pneumophila populations in environmental water is very important for public health, because the bacterium is the primary cause of Legionnaires' disease. The conventional plate culture method, similar to ISO 11731 (2017) , is generally used to enumerate Legionella spp. in water samples. The method, however, is highly complicated and requires sample handling and colony identification skills due to the growth of non-target bacteria and fungi on the agar plates. Thus, the development of a simpler method that could deliver accurate enumeration results is urgently needed.The Legiolert ® / Quanti-Tray ® most probable number (MPN) method (IDEXX Laboratories, USA) is a novel method to enumerate L. pneumophila in potable (e.g., tap water) and non-potable (e.g., cooling tower water) water samples. According to the manufacturer's instructions, the principle of this method is based on a bacterial enzyme detection technology that signals the presence of L. pneumophila through utilization of a substrate present in the Legiolert reagent. L. pneumophila cells grow rapidly and reproduce using the rich supply of nutrients present in the Legiolert reagent. Actively growing strains of L. pneumophila use the added substrate to produce a brown color indicator. There are four different protocols in the manufacturer's instructions, two for potable water and the other two for non-potable water. Recent papers have reported method comparison studies for the enumeration of L. pneumophila between Legiolert and the conventional plate culture method from potable water, such as hot water taps, showers, and ice machines (Sartory et al., 2017;Petrisek and Hall, 2017;Spies et al., 2018) and non-potable water, such as cooling tower water (Petrisek and Hall, 2017;Rech et al., 2018) . In this paper, we report the first comparison of the enumeration of L. pneumophila from bath water samples using the two Legiolert potable water methods and the conventional plate culture method.All water samples were collected from natural hot springs (53 samples) and public baths using tap water (79 samples) between May 2018 and March 2019. These samples were collected in sterile 500-mL polypropylene bottles with sodium thiosulfate, and were examined within 3 d after sample collection.The Legiolert/Quanti-Tray MPN method was carried out according to manufacturer's instructions. We compared the two potable water protocols (10 mL and 100 mL protocols) described in the instructions. In the 10 mL protocol (Legiolert-10 mL) , 10 mL of each water sample was transferred into a sterile 120 mL vessel and diluted with 90 mL of sterile deionized water. In the 100

show abstract

mentioning

confidence: 99%

Evaluation of Legiolert for Quantification of <i>Legionella pneumophila</i> from Bath Water Samples

Inoue¹,

Baba²,

Tayama³

2020

Biocontrol Sci.

View full text Add to dashboard Cite

show abstract

“…The defects of the bb0562 mutant clones were the most pronounced for spirochetes in mid-log and early stationary phase ( S5 Fig ). Recently, bacterial colony formation in solid media has been linked to fatty acid availability [ 37 ]. Because the B .…”

Section: Resultsmentioning

confidence: 99%

“…E . coli lacking the fabA gene, which is essential for biosynthesis of long chain unsaturated fatty acids, is highly attenuated for colony formation on solid media under fatty acid limited conditions [ 37 ]. However, this phenotype is less pronounced for growth in liquid media, suggesting that fatty acids are particularly important for bacterial colony formation in vitro [ 37 ].…”

Section: Discussionmentioning

confidence: 99%

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments

et al. 2021

View full text Add to dashboard Cite

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

show abstract

“…The strongest impact might be through the change from liquid to solid cultivation conditions. Large differences in culturability for several known strains and isolation campaigns have been described depending on the choice of liquid or solid cultivation media [51][52][53].…”

Section: Discussionmentioning

confidence: 99%

Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities

Mahler

Niehs

Martin

et al. 2021

eLife

View full text Add to dashboard Cite

Antibiotics from few culturable microorganisms have saved millions of lives since the 20th century. But with resistance formation, these compounds become increasingly ineffective, while the majority of microbial and with that chemical compound diversity remains inaccessible for cultivation and exploration. Culturing recalcitrant bacteria is a stochastic process. But conventional methods are limited to low throughput. By increasing (i) throughput and (ii) sensitivity by miniaturization, we innovate microbiological cultivation to comply with biological stochasticity. Here, we introduce a droplet-based microscale-cultivation system, which is directly coupled to a high-throughput screening for antimicrobial activity prior to strain isolation. We demonstrate that highly parallelized in-droplet cultivation starting from single cells results in the cultivation of yet uncultured species and a significantly higher bacterial diversity than standard agar plate cultivation. Strains able to inhibit intact reporter strains were isolated from the system. A variety of antimicrobial compounds were detected for a selected potent antibiotic producer.

show abstract

Isolation of colonization-defective Escherichia coli mutants reveals critical requirement for fatty acids in bacterial colony formation

Cited by 13 publications

References 57 publications

Evaluation of Legiolert for Quantification of <i>Legionella pneumophila</i> from Bath Water Samples

Evaluation of Legiolert for Quantification of <i>Legionella pneumophila</i> from Bath Water Samples

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments

Highly parallelized droplet cultivation and prioritization of antibiotic producers from natural microbial communities

Contact Info

Product

Resources

About