Endemic transmission of nosocomial pathogens, especially gram-negative organisms, in a hospital environment is often poorly defined and difficult to control. While some studies have demonstrated that previous occupancy of an ICU room by a patient with multidrug-resistant (MDR) gram-negative bacteria is a risk factor for acquisition by subsequent occupants, others have infrequently recovered gram-negatives from the hospital environment
[26, 27]. Additionally, while contact precautions have been demonstrated to have efficacy in control of transmission of MRSA, VRE and C. difficile, the evidence is less robust for gram-negative organisms
[28, 29]. Although gram-negatives are the predominant pathogens in cases of ventilator-associated pneumonia and in ICU HAIs, where death from HAI is most likely to occur, existing guidelines pertaining to control of MDR pathogens either exclude gram-negatives or acknowledge limitations in recommendations pertaining to these organisms
[30–32]. Increased ability to detect environmental reservoirs of these organisms should lead to improvements in targeted control efforts. Prior studies have evaluated (by TCM) the frequency of microorganisms on high-use surfaces, HCW, PPE, and other items in the healthcare environment, with widely differing results depending on the site sampled and organism of interest. Many have focused on one organism in the immediate surroundings of a patient known to be colonized with that organism
[3, 9, 26, 33–35]. Apart from this context, studies in non-outbreak settings often evaluate epidemiologically significant pathogens for one site of interest per study. One evaluation of MDR A. baumannii contamination in a medical intensive care unit found none except in colonized patients’ immediate surroundings
. In our institution, an evaluation (by TCM) was made of protective lead garments at various sites, with only 5 of 182 samples positive for any bacteria, all normal skin flora; another assessment of computer keyboards/mice recovered S. aureus, Acinetobacter spp., or Pseudomonas spp. on 17% of tested surfaces
[2, 4]. One study demonstrated a majority of bedside charts in the ICU were contaminated with MDR bacteria
Against this backdrop, we sought to determine the burden and spectrum of microorganisms detected from HCW, PPE, and a variety of high-use hospital environmental surfaces by PCR/ESI-TOF-MS compared to TCM, and to compare the two methodologies in reference to the most commonly identified microorganisms detected among clinical cultures. This study is the first to our knowledge to evaluate selected sections of the hospital microbiome, comparing TCM and the unbiased T-5000-based PCR/ESI-TOF-MS method, in an effort to understand potential reservoirs of endemic nosocomial bacteria. Compared to traditional culture, PCR/ESI-TOF-MS detected more microbes, including more pathogens of potential clinical relevance, from a greater number of surfaces, hands of HCW, and PPE. PCR/ESI-TOF-MS also disproportionately recovered more gram-negative organisms missed by culture than S. aureus. As would be expected, both TCM and PCR/ESI-TOF-MS also detected many clinically less important organisms. Potentially clinically relevant pathogens accounted for similar proportions of all results (an estimated “signal-to-noise” ratio), unless streptococci, which are not typically considered major HAI pathogens, were included. However, compared to TCM, PCR/ESI-TOF-MS was able to detect potentially clinically relevant pathogens from 3-6x the number of sites screened, depending upon the inclusivity of the definition of “clinically relevant”. There is no standard definition for potential clinical relevance, and lower-virulence organisms, such as coagulase-negative staphylococci, can present major problems for patients with orthopedic or other implanted devices, or severely compromised patients. The study definition was chosen in order to reflect the most common pathogens seen clinically at the time and to include organisms for which there is high concern for virulence and/or drug resistance
[32, 37]. Additionally, much greater microorganism diversity was detected by PCR/ESI-TOF-MS, including unexpected organisms with high virulence or outbreak potential (e.g. Clostridium tetani, S. pyogenes). All surfaces, hands, and PPE samples demonstrated large numbers of recoverable pathogens, but without clear trends related to number of organisms by site. Based on these data, no obvious target for increased infection control efforts was seen in the study units. As this study was designed as a pilot study with a small number of sites/samples tested, there are clear limitations to the generalizability of the results to an entire hospital microbiome. It is also difficult to draw conclusions about organisms such as Shigella flexneri and Borrelia turicatae recovered from the hospital environment in the absence of known clinical cases during the study period. It is possible that these represent misidentifications of related organisms, as with hundreds of identified organisms, even 99% specificity would lead to several misidentifications. However, previous characterizations of this technology with 405 unique bacterial species have demonstrated accurate characterization in 95% of instances, with the remaining 5% unresolved species all accurate to the genus level
. Other limitations of the use of PCR/ESI-TOF-MS in this context include the possibility of detection of nonviable organisms, cost, the semi-quantitative nature of the data, and inability to recover specific strains linked to a patient or outbreak isolate. In this study, PCR/ESI-TOF-MS was not used to detect specific strains of bacteria detected, though it can be and has been used specifically for rapid genotyping of A. baumannii, S. aureus and Streptococcus pneumoniae. Thus, this technology may yet prove useful in outbreak investigations using environmental sampling, especially since it detected 4–5 fold higher numbers of pathogens per site without adversely affecting the ratio of clinically irrelevant microbes.
Interestingly, PCR/ESI-TOF-MS detected widespread resistance elements throughout the hospital environment. In the case of mecA, most were associated with CNS codetections rather than S. aureus. As mecA elements are widely distributed in CNS, this is not surprising
. VanA was not detected with enterococcus by PCR/ESI-TOF-MS, but did have an association with the presence of Lactobacillus spp. While this organism is largely intrinsically resistant to vancomycin, previous studies have demonstrated that this is not related to the presence of vanA, which to our knowledge has not been demonstrated in lactobacilli
[40, 41]. It is interesting that one of the samples positive for vanA, but without enterococci by PCR/ESI-TOF-MS, grew a susceptible Enterococcus spp. on clinical culture. Furthermore, none of the five samples positive for enterococci by TCM had this organism codetected by PCR/ESI-TOF-MS, generating a question of whether there might have been specific Enterococcus spp. detection problems, which have not been previously described. Overall, there were no attempts to resolve discordant results from TCM and PCR/ESI-TOF-MS, given that essentially every sample showed discordance, at least in greater number of pathogens isolated by the latter method. The PCR/ESI-TOF-MS technology does not screen for all possible resistance genes, and as applied here only detects the resistance element, without reporting whether it is incorporated into the genome of an organism. If more than one organism is present in the test sample along with the resistance element, it was not clear with which organism the element might be associated, if any. It is possible that free-floating or promiscuous plasmids are responsible for some of these detections, which also has significance for infection transmission in recent literature
. Given the difficulty of performing plasmid genetic analysis and whole genome sequencing of unculturable organisms, it is likely that horizontal transmission and intergenus transfer of antimicrobial resistance elements plays a larger role in healthcare-associated transmission of gram-negative MDR pathogens than has yet been described
. The ability of PCR/ESI-TOF-MS to screen for a broad spectrum of genetic elements may be a starting point for hypothesis development related to horizontal transmission.