Spatial Characteristics of the Efflux Pump MexB Determine Inhibitor Binding.

The multidrug efflux transporters MexB and MexY in Pseudomonas aeruginosa and AcrB in Escherichia coli contribute to these organisms' multidrug resistance. Efflux pump inhibitor (EPI) ABI-PP inhibits MexB and AcrB, but not MexY. We previously determined the structure of ABI-PP bound to the hydrophobic trap (the inhibitor-binding pit) of AcrB and MexB. The insensitivity of MexY to ABI-PP was attributed to a bulky tryptophan (Trp). AcrB(Phe178Trp) became uninhibited by ABI-PP, while MexY(Trp177Phe) resensitized MexY for ABI-PP. Interestingly, ABI-PP was able to inhibit MexB(Phe178Trp). Thus, it is not clear which bulky amino acid mutations are critical for inhibitor binding in MexB. Here, we investigated the pit of MexB in more detail, and elucidated which Trp mutation locations in the pit were hindering ABI-PP binding, but did not affect the function of the efflux pumps. Mutating positions 139, 277, 279, and 612 to tryptophan eliminated the inhibitory effect. However, the tryptophan mutation at position 571 did not cause any effect. These results show that the effectiveness of EPIs is greatly affected by mutations in different locations, and that binding of EPIs is partly attributed by spatial characteristics. These results should be taken into account for new inhibitor and drug discovery.

Identification of Genetic Variants via Bacterial Respiration Gas Analysis.

Indole is a signal molecule derived from the conversion of tryptophan, and it is present in bacterial respiratory gas. Besides influencing bacterial growth, indole exhibits effects on human health, including a positive effect on inflammation and protection against pathogens. However, a high fecal indole concentration (FIC) can suggest an unbalanced gut flora or the presence of certain pathogens. To analyze the indole produced by bacteria, its collection and detection is required. Traditional methods usually require centrifugation of liquid bacterial culture medium and subsequent extraction of indole from the medium or partial purification of indole from fecal samples (e.g., by distillation or extraction). In this study, we demonstrate the possibility of identifying gas contents directly from bacteria, and we distinguish the difference in species and their genetics without the need to centrifuge or extract. Using an absorbent sheet placed above a liquid culture, we were able to collect gas content directly from bacteria. Gas chromatography-mass spectrometry (GC-MS) was used for the analysis. The GC-MS results showed a clear peak attributed to indole for wild-type Escherichia coli cells (MG1655 and MC4100 strains), whereas the indole peak was absent in the chromatograms of cells where proteins, part of the indole production pathway from tryptophan (TnaA and TnaB), were not expressed (by using tnaAB-deleted cells). The indole observed was measured to be present in a low nmol-range. This method can distinguish whether the bacterial genome contains the tnaAB gene or not and can be used to collect gas compounds from bacterial cultures quickly and easily. This method is useful for other goals and future research, such as for measurements in restrooms, for food-handling facilities, and for various applications in medical settings.

A large hepatic cyst with obstructive jaundice successfully treated with single-incision laparoscopic deroofing.

We herein present a case of hepatic cysts causing obstructive jaundice that was treated with single-incision laparoscopic deroofing. A 72-year-old female patient was referred to hospital due to a large hepatic cyst that compressed the intrahepatic bile ducts. The patient was scheduled to undergo single-incision laparoscopic deroofing. The EZ ACCESS(TM) oval type (Hakko Co. Ltd.) was placed at the umbilicus using a 25-mm incision with two 5-mm trocars. An additional 12-mm port was placed at the left epigastric region. We unroofed and excised the cyst wall using a vessel sealing system in liver segment 4. After surgery, the patient was found to be asymptomatic. The unroofed cysts were completely diminished. Notably, the remnant liver had fairly regenerated. The estimated regeneration volume of the normal liver was 153 cm(3). To prevent surgical complications, clinicians should perform adequate management and use of devices. To prevent postoperative recurrence of cysts, performing complete deroofing is essential. Single-incision laparoscopic deroofing contributes to improving the quality of life of patients and should be considered a standard treatment.