Development of a sensor for disulfide bond formation in diverse bacteria.

In bacteria, disulfide bonds contribute to the folding and stability of proteins important for processes in the cellular envelope. In Escherichia coli, disulfide bond formation is catalyzed by DsbA and DsbB enzymes. DsbA is a periplasmic protein that catalyzes disulfide bond formation in substrate proteins, while DsbB is an inner membrane protein that transfers electrons from DsbA to quinones, thereby regenerating the DsbA active state. Actinobacteria including mycobacteria use an alternative enzyme named VKOR, which performs the same function as DsbB. Disulfide bond formation enzymes, DsbA and DsbB/VKOR, represent novel drug targets because their inhibition could simultaneously affect the folding of several cell envelope proteins including virulence factors, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. We have previously developed a cell-based and target-based assay to identify molecules that inhibit the DsbB and VKOR in pathogenic bacteria, using E. coli cells expressing a periplasmic ß-Galactosidase sensor (ß-Galdbs), which is only active when disulfide bond formation is inhibited. Here, we report the construction of plasmids that allows fine-tuning of the expression of the ß-Galdbs sensor and can be mobilized into other gram-negative organisms. As an example, when expressed in Pseudomonas aeruginosa UCBPP-PA14, which harbors two DsbB homologs, ß-Galdbs behaves similarly as in E. coli, and the biosensor responds to the inhibition of the two DsbB proteins. Thus, these ß-Galdbs reporter plasmids provide a basis to identify novel inhibitors of DsbA and DsbB/VKOR in multidrug-resistant gram-negative pathogens and to further study oxidative protein folding in diverse gram-negative bacteria. IMPORTANCE: Disulfide bonds contribute to the folding and stability of proteins in the bacterial cell envelope. Disulfide bond-forming enzymes represent new drug targets against multidrug-resistant bacteria because inactivation of this process would simultaneously affect several proteins in the cell envelope, including virulence factors, toxins, proteins involved in outer membrane biogenesis, cell division, and antibiotic resistance. Identifying the enzymes involved in disulfide bond formation in gram-negative pathogens as well as their inhibitors can contribute to the much-needed antibacterial innovation. In this work, we developed sensors of disulfide bond formation for gram-negative bacteria. These tools will enable the study of disulfide bond formation and the identification of inhibitors for this crucial process in diverse gram-negative pathogens.

Methicillin-resistant Staphylococcus aureus contamination of high-touched surfaces in a university campus.

AIM: Methicillin-resistant Staphylococcus (MRSA) is a public and occupational health concern, both in community and healthcare settings. In recent years, community-acquired MRSA (CA-MRSA) has emerged as a major causative agent of infections in individuals with no health care exposure or any of the classical risk factors associated with infections. Environmental surfaces frequently touched by hands play a role in the transmission of CA-MRSA, where inanimate objects are considered potential reservoirs and the source of MRSA infections. The purpose of this study was to examine the prevalence of MRSA on environmental surfaces inside a university campus. METHODS AND RESULTS: A total of 1078 high-touch surface samples were collected from door handles, light switches, desks, keyboards and restroom surfaces. MRSA isolates were identified and confirmed by PCR, utilizing the Staph. aureus nuc and mecA genes. Antibiotic resistance profiles were determined using disc diffusion and minimum inhibitory concertation methods. In addition, the ability to form biofilms was investigated by the 96-well plate microdilution technique. PCR assays were performed to detect enterotoxin and antibiotic-resistant genes. The genetic diversity of MRSA was determined through multi-locus sequence typing (MLST), spa and agr typing methods. The overall contamination of Staph. aureus and MRSA was 14.6% (157/1078) and 2.8% (30/1078), respectively. The highest rate of MRSA contamination was detected in restroom sinks and door handles. All MRSA isolates were MDR, with the highest resistance observed was against trimethoprim-sulfamethoxazole. Most MRSA isolates (29/30, 97%) carried at least one gene encoding for staphylococcal enterotoxins (SE), with 10 different SE genotypes were observed. A total of 16 different spa types were detected among the 30 MRSA isolates. Multi-locus sequence typing revealed that 21 MRSA isolates belonged to eight known sequence types (ST), while nine isolates were novel strains. The most detected ST and spa types were ST22 and t223, respectively. Agr types I and III were represented in 28 out of the 30 isolates. The majority of the isolates carried SCCmec type IV, but only one isolate was positive for PVL. CONCLUSIONS: Our findings signify the potential of the high-touch surfaces in harbouring and transmitting MRSA to campus staff and students. Thus, the implementation of effective prevention measures outside the healthcare setting is needed to reduce the risk of acquiring CA-MRSA infections. SIGNIFICANCE AND IMPACT: MRSA infections impose a profound economic burden due to illness and productivity loss. The results of this study not only help us to better understand the environmental reservoirs of this pathogen, but also provide information about its transmission pathways and healthcare settings entry routs.

Isolation of extensively drug resistant Acinetobacter baumannii from environmental surfaces inside intensive care units.

BACKGROUND: Acinetobacter baumannii is a nosocomial pathogen that has emerged as a major threat in the health-care settings, particularly intensive care units (ICUs). The aim of this study was to investigate the prevalence of A. baumannii in the environment of intensive care and emergency units in 4 hospitals in Jordan. METHODS: A total of 311 surface and 26 air samples were collected from 6 different ICUs and 2 emergency units. Examined high-touch surfaces included bed rails, sinks, food tables, trolley handles, ventilator inlets, blankets, sheets, door handles, light switches, bedside tables and drawers, curtains, normal saline stands and neonatal incubators. A. baumannii isolates were identified by CHROMagar and confirmed using 2 different PCR assays. All obtained isolates were characterized for their antibiotic resistance phenotypes, biofilm formation capacities and were typed by multi-locus sequence typing. RESULTS: Of the 337 samples, 24 A. baumannii isolates were recovered, mostly from surfaces in the internal medicine ICUs. Among the 24 isolates, 10 isolates were classified as extensively-resistant (XDR), harbored the blaOXA-23 like gene and able to form biofilms with varying capacities. ST2 was the most frequent sequence type, with all ST2 isolates classified as XDRs. CONCLUSIONS: Our results showed that high-touch surfaces of adult and pediatric ICUs were contaminated with XDR A. baumannii isolates. Therefore, the cleaning practices of the surfaces and equipment surrounding ICU patients should be optimized, and health-care workers should continuously wash their hands and change their gloves constantly to control the spread of this pathogen.