The use of PCR/Electrospray Ionization-Time-of-Flight-Mass Spectrometry (PCR/ESI-TOF-MS) to detect bacterial and fungal colonization in healthy military service members

One hundred one healthy active duty service members presenting to the outpatient Troop Medical Clinic for acute care at Fort Sam Houston, TX were enrolled. Study participants were 18 years and older who had no overseas travel or deployment within the past 6 months, no antibiotic use in the preceding 30 days, and were being seen for non-infectious medical conditions. Written informed consent was obtained from study participants upon enrollment. Individuals were excluded from the study if they were deemed to have an acute or chronic infection that may have altered normal bacterial flora or that involved a proposed anatomic sample site. The study protocol was approved by the San Antonio Military Medical Center (SAMMC) Institutional Review Board. The results presented here are a sub-study of a larger protocol examining various methods for detecting microbial colonization in healthy US military personnel; initial results on methicillin-resistant and methicillin-sensitive Staphylococcus aureus (MRSA and MSSA) and multidrug-resistant (MDR) GNB colonization have been published [14, 15]. Our current study further examines bacterial and fungal colonization of healthy U.S. military individuals, including the correlation of GNB detection by PCR/ESI-TOF-MS and traditional culture.

Individuals were screened with culture swabs (Copan Stuart liquid media culture, Copan Inc., Brescia, Italy) from various anatomic sites (nares, oropharynx, and groin). Each participant was sampled by application of the swab in a repetitive twisting motion at each anatomic site, firmly brushing the skin or mucosa to ensure transfer of cells onto the swab tip. Samples for PCR/ESI-TOF-MS were frozen at -20^o C or lower and transported on dry ice for batched testing to the Center for Genomic Sciences, Allegheny Singer Research Institute, Pittsburgh, PA, USA. A separate set of swabs collected in the same fashion were transported to the SAMMC research microbiology laboratory and underwent immediate processing for microbial culture to identify GNB. The analysis of coagulase-negative Staphylococcus (CNS) and S. aureus detection by traditional culture and PCR/ESI-TOF-MS methods has been published elsewhere [14]. We did not evaluate other gram-positive bacteria, fungus or anaerobes by traditional culture due to cost constraints.

Frozen swabs were thawed after transport, placed in microcentrifuge tubes containing 270 μl of ATL Lysis buffer (Qiagen, Germantown, MD, cat# 19076) and 30 μl proteinase K (Qiagen, cat# 19131) and then incubated at 56 °C for 1 h. Next, 100 μl of a mixture containing 50 μl each of 0.1 mm and 0.7 mm Zirconia beads (Biospec, Bartlesville, OKcat# 11079101z, 11079107zx, respectively) were added to the samples which were then homogenized for 10 min at 25 Hz using a Qiagen Tissuelyser. Nucleic acid from the lysed sample was then extracted using the Qiagen DNeasy kit (Qiagen cat# 69506). 10 μl of each sample was loaded per well onto the BAC detection PCR plate (Abbott Molecular, Carlsbad, CA cat# PN 05 N13-01). The BAC detection plate contains 96 wells each with 16 primers that survey all bacterial organisms by using multiple omnipresent loci (e.g., 16S rDNA sequences) and multiple pluripresent loci (e.g., the tufB gene). The BAC assay has been validated against 613 organisms [9]^. The system also detects the presence of several key antibiotic resistance markers: vanA and vanB (vancomycin resistance) in Enterococcus spp., KPC-3 (carbapenem resistance) in GNB, and mecA (methicillin resistance) in Staphylococcus spp. An internal calibrant of synthetic nucleic acid template is also included in each assay, controlling for false negatives (e.g., from PCR inhibitors) and enabling a semi-quantitative analysis of the amount of template DNA present. PCR amplification was performed according to Ecker et al. [9] The PCR products were then desalted in a 96-well plate format and sequentially electrosprayed into a time-of-flight mass spectrometer. The spectral signals were processed to determine the masses of each of the PCR products present with sufficient accuracy to determine the base composition of each amplicon. Using combined base compositions from multiple PCRs, the identities of the pathogens and a semi-quantitative determination of their relative concentrations in the starting sample were established by using a proprietary algorithm which interfaces with the Ibis database of known organisms.

All isolates were plated onto both Trypticase^™ Soy Agar with 5 % sheep blood (sheep blood agar, BBL, Cockeysville, MD, USA) and MacConkey agar (BBL, Cockeysville, MD, USA) in order to isolate aerobic GNB colonies. Colonies grown on MacConkey agar and colonies consistent with gram-negative morphology on sheep blood agar were sub-cultured onto sheep blood agar to assure culture purity and then frozen at -80^o C for subsequent evaluation. Frozen isolates underwent two passages on sheep blood agar, followed by automated testing for species-identification and susceptibility via the BD Phoenix Automated Microbiology System (Becton Dickinson and Company, Franklin Lakes, NJ) using NMIC/ID-123 panels according to manufacturer’s guidelines.

Organisms were categorized based on taxonomy and morphological features. Basic descriptive statistics were used to summarize the findings using SPSS (IBM®SPSS®Statistics Version 19). Comparison between PCR/ESI-TOF-MS and traditional culture results was only performed on GNB utilizing Spearman’s rank correlation.

Three anatomic sites (nares, oropharynx, and groin) of 101 participants were swabbed, with one subject declining the oropharynx swab. The median age of participants was 23 [IQR 22, 23] years, with 69 % being male.

PCR/ESI-TOF-MS identified at least one organism in every subject, with a mean number of 11.8 [SD 2.8] organisms per subject. The most commonly deteceted gram positive and gram negative bacteria, by anatomic site, are shown in Figs. 1 and 2. The oropharynx site yielded the highest mean number of organisms, 4.2 [SD 2.0] followed by groin 3.8 [SD 1.4] and nares 3.8 [SD 1.3] (Table 1).

Overall, aerobic gram-positive bacteria were more prevalent than GNB (870 vs. 140 total isolates, respectively) and were detected from every subject, with a mean number of 8.6 [SD 2.0] organisms, the highest number being detected from groin swabs, 3.3 [SD 3.3] followed by nares 2.7 [SD 1.3] and oropharynx 2.6 [SD 1.3] (Table 1). PCR/ESI-TOF-MS identified 870 aerobic gram-positive bacteria (329 from groin swabs, 275 from nares, and 266 from oropharynx). The most common organisms (341) were CNS with numerous other gram-positive bacteria noted (Table 2). The mecA gene was identified in nearly every subject (96). No resistance elements of vanA and vanB were detected in this study.

Aerobic GNB were also commonly found by PCR/ESI-TOF-MS in 80 subjects, with a mean number of 1.4 [SD 1.0] organisms, primarily the oropharynx followed by nares and groin (Table 1). In total, 140 aerobic GNB were identified (94 from oropharynx swabs, 32 from nares, and 14 from groin). The most common GNB identified were Neisseria spp. (59), followed by Haemophilus influenzae (21), Moraxella spp. (19), and Escherichia coli (7) (Table 3). As was seen with gram-positive bacteria, several GNB known to cause human disease were frequently found (Table 3). No genes commonly associated with GNB resistance were detected by PCR/ESI-TOF-MS.

Multiple anaerobic bacteria were detected by PCR/ESI-TOF-MS in 75 subjects, with a mean number of 1.2 [SD 0.98] organisms; the most common site being the nares (56 subjects – mean number of 0.6 [SD 0.6] organisms) (Table 1). In total, 96 anaerobic gram-positive bacteria (6 from oropharynx swabs, 57 from nares, and 33 from groin) and 27 anaerobic GNB were identified (23 from oropharynx swabs, 3 from nares, and 1 from groin). Propionibacterium acnes was the most commonly identified anaerobic gram-positive bacteria (70) (Table 4).

PCR/ESI-TOF-MS identified fungal species in 46 subjects with a mean of 0.60 [SD 0.79] organisms per individual (Table 1). A total of 61 fungal organisms, with 39 in the oropharynx, 14 nares, and 8 groin were detected (Table 5).

Traditional culture identified colonization of 34 sites from 23 subjects with GNB. The most commonly recovered organism was Pseudomonas aeruginosa with 4 isolates, all of which were from oropharyngeal sites. Other GNB included E.coli (3 isolates), K. pneumoniae (3 isolates), and Serratia marcescens (3 isolates). A complete list of less common GNB is shown in Table 6. Traditional culture also did not detect any MDR GNB.