Optical Mapping: Detecting Genomic Resistance Cassettes in MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant bacterium with a global presence in healthcare facilities as well as community settings. The resistance of MRSA to beta-lactam antibiotics can be attributed to a mobile genetic element called the staphylococcal cassette chromosome mec (SCCmec), ranging from 23 to 68 kilobase pairs in length. The mec gene complex contained in SCCmec allows MRSA to survive in the presence of penicillin and other beta-lactam antibiotics. We demonstrate that optical mapping (OM) is able to identify the bacterium as S. aureus, followed by an investigation of the presence of kilobase pair range SCCmec elements by examining the associated OM-generated barcode patterns. By employing OM as an alternative to traditional DNA sequencing, we showcase its potential for the detection of complex genetic elements such as SCCmec in MRSA. This approach holds promise for enhancing our understanding of antibiotic resistance mechanisms and facilitating the development of targeted interventions against MRSA infections.

Restriction endonucleases from invasive Neisseria gonorrhoeae cause double-strand breaks and distort mitosis in epithelial cells during infection.

The host epithelium is both a barrier against, and the target for microbial infections. Maintaining regulated cell growth ensures an intact protective layer towards microbial-induced cellular damage. Neisseria gonorrhoeae infections disrupt host cell cycle regulation machinery and the infection causes DNA double strand breaks that delay progression through the G2/M phase. We show that intracellular gonococci upregulate and release restriction endonucleases that enter the nucleus and damage human chromosomal DNA. Bacterial lysates containing restriction endonucleases were able to fragment genomic DNA as detected by PFGE. Lysates were also microinjected into the cytoplasm of cells in interphase and after 20 h, DNA double strand breaks were identified by 53BP1 staining. In addition, by using live-cell microscopy and NHS-ester stained live gonococci we visualized the subcellular location of the bacteria upon mitosis. Infected cells show dysregulation of the spindle assembly checkpoint proteins MAD1 and MAD2, impaired and prolonged M-phase, nuclear swelling, micronuclei formation and chromosomal instability. These data highlight basic molecular functions of how gonococcal infections affect host cell cycle regulation, cause DNA double strand breaks and predispose cellular malignancies.

Lactobacillus decelerates cervical epithelial cell cycle progression.

We investigated cell cycle progression in epithelial cervical ME-180 cells during colonization of three different Lactobacillus species utilizing live cell microscopy, bromodeoxyuridine incorporation assays, and flow cytometry. The colonization of these ME-180 cells by L. rhamnosus and L. reuteri, originating from human gastric epithelia and saliva, respectively, was shown to reduce cell cycle progression and to cause host cells to accumulate in the G1 phase of the cell cycle. The G1 phase accumulation in L. rhamnosus-colonized cells was accompanied by the up-regulation and nuclear accumulation of p21. By contrast, the vaginal isolate L. crispatus did not affect cell cycle progression. Furthermore, both the supernatants from the lactic acid-producing L. rhamnosus colonies and lactic acid added to cell culture media were able to reduce the proliferation of ME-180 cells. In this study, we reveal the diversity of the Lactobacillus species to affect host cell cycle progression and demonstrate that L. rhamnosus and L. reuteri exert anti-proliferative effects on human cervical carcinoma cells.