These days, patients with suspected nosocomial infections are assessed by traditional culture methods, and a positive result leads to appropriate antibiotic therapy. Routine screening for methicillin-resistant Staphylococcus aureus (MRSA), however, only occurs at admission. With the increase in need for patient isolation and long-term in-hospital patient management, hospitals are seeking a benchmark assay that could generate relevant evidence for implementing an appropriate intervention program as early as possible.
There are key genetic factors such as antibiotic-resistance genes and toxin genes that make a resident microbe a nosocomial pathogen. Corresponding variants of these genes may increase the pathogenicity, as reflected in the minimum inhibitory concentration (MIC) value. Most of these genetic markers are carried by mobile genetic materials (plasmids and transposons) and hence are transferable across species barriers.
Furthermore, based on selection pressure (e.g., antibiotic usage), the incidence and genetic makeup of these markers will vary but they can also be purged from the carrier organism in the absence of selection pressure.
The genes carried by the Staphyloccus chromosome cassette (SSCmec) vary, and mere identification of the cassette itself may not provide information about the genes it carries nor their variations. Therefore, the key deciding factors for monitoring nosocomial infections are the nosocomial-associated genes themselves and their respective variants, rather than the carrier vehicles such as organisms, plasmids, transposons, or genetic cassettes.
Although there are methods to type the carrier organisms, they seldom include identifying the genes that are associated with nosocomial infections. Thus, there is a need to periodically characterize selected nosocomial-associated genes to monitor their changes and spread in the local microbial gene pool.
Although the need for intervention programs is well recognized, the lack of appropriate technology has prevented implementation at most healthcare facilities. In this article, we describe the application of a multiplex-sequencing technology that can be tailored to generate data on relevant microbial genetic factors and thereby facilitate implementation of an evidence-based surveillance program.
The data presented here was simultaneously generated to identify the methicillin-resistance marker (mecA), markers that distinguish community-acquired from hospital-acquired infections (mecR1 and mec1), and a marker (spa) for source identification. This assay not only accurately identifies methicillin resistance, it also sub-types the MRSA as community acquired (CA-MRSA) or hospital acquired (HA-MRSA). In addition, the hyper-variable region of the spa gene can be used for tracking and source identification.
Multiplex DNA Sequencing
MultiGen Diagnostics’ MultiGEN technology allows simultaneous sequencing of multiple amplicons generated either from the same genome, different genomes, or a combination of both.
In this assay we used three relevant SCC-associated gene segments (Pb2´, mecR1, and mec1) and a region (spa) of the S. aureus genome.
The MultiGEN process includes the preparation of total nucleic acid, followed by amplification using four PCR primer pairs to produce four amplicons that are simultaneously sequenced at the 3´end. Expected read sequences of the genes are shown in the Figure. The assay was developed using S. aureus genomic DNA, which was later used as a positive control in the analysis of the isolates.
Twenty five clinical isolates on agar slants were obtained from the clinical laboratory of a local hospital. Bacterial colonies were picked and suspended in 200 µL of sterile water. Total DNA was extracted using the DNA Mini Kit.
All four genes (Pb2´, mec1, mecR1, and spa) were amplified using the MultiGEN-MRSA Amp kit. In a 50 µL reaction volume, 3 µL of the DNA extract was amplified with 5 units of Taq polymerase for 35 cycles. Amplicons were purified using Ampure. All four amplicons were simultaneously sequenced by cycle sequencing.
Inincorporated dye terminators were removed using Cleanseq. Then, 35 µL of eluate was freeze-dried in a speed-vac and resuspended in 10 µL of Hi-Di foramide. Samples were then analyzed by capillary electrophoresis. They were drawn into the capillary by electrokinetic injection at 2 Kv for 12 seconds, and electrophoresis was carried out at 15 Kv for 20 minutes.
The nucleotide sequences generated were verified by BLAST search to confirm the authenticity of sequences generated from the isolates. The assays were carried out with negative controls and genomic DNA from S. aureus was used as positive control.
The process from extraction to generating nucleotide sequences took less than eight hours. Our initial panel consists of the following genetic markers: SCCmec that expresses the PBP2´ gene (mec A), accessory genes (membrane-bound transducer gene mecR1 that derepresses the repressor gene mec1) that control the expression of PBP2´, and the hypervariable spa region.