Abstract
We present extremity wound microbiology data from 250 combat casualties (2009–2012). Confirmed extremity wound infections (EWIs) were based on clinical and laboratory findings. Suspected EWIs had isolation of organisms from wound cultures with associated signs/symptoms not meeting clinical diagnostic criteria. Colonized wounds had organisms isolated without any infection suspicion. A total of 335 confirmed EWIs (131 monomicrobial and 204 polymicrobial) were assessed. Gram-negative bacteria were predominant (57% and 86% of monomicrobial and polymicrobial infections, respectively). In polymicrobial infections, 61% grew only bacteria, while 30% isolated bacteria and mold. Multidrug resistance was observed in 32% of isolates from first monomicrobial EWIs ±3 days of diagnosis, while it was 44% of isolates from polymicrobial EWIs. Approximately 96% and 52% of the suspected and colonized wounds, respectively, shared ≥1 organism in common with the confirmed EWI on the same patient. Understanding of combat-related EWIs can lead to improvements in combat casualty care.
Keywords: extremity wounds, wound microbiology, trauma-related infections, extremity infections, open fractures
1.0. INTRODUCTION
Extremity wounds are prevalent among combat casualties and often lead to infectious complications, resulting in significant morbidity and mortality (Brown et al., 2010; Murray, 2008; Murray et al., 2001; Tribble et al., 2011). During the wars in Iraq and Afghanistan, multidrug-resistant organisms (MDROs) were frequently associated with wound colonization and trauma-related infections (Hospenthal et al., 2011; Keen et al., 2010; Murray et al., 2006, 2011; Weintrob et al., 2013), presenting further challenges for clinicians and potentially impacting patient outcomes.
Although microbiology of extremity wounds sustained during combat has been examined (Calhoun et al., 2008; Murray et al., 2006; Petersen et al., 2007; Wallum et al., 2015; Yun et al., 2006), prior studies frequently involved a small population, only considered initial cultures, and did not include information on infections. Understanding the complicated wound microbiome is an essential element of improving trauma patient care. Therefore, we describe the microbiology of combat-related extremity wounds and wound infections, over a three-year period.
2.0. METHODS
2.1. Study Population and Design
Data were collected through the Department of Defense (DoD) - Department of Veterans Affairs Trauma Infectious Disease Outcomes Study (TIDOS) (Tribble et al., 2011), an observational, longitudinal, multicenter study of short- and long-term infectious complications following deployment-related trauma. Patients were eligible for inclusion if they sustained a combat-related extremity injury in Iraq or Afghanistan (June 1, 2009 - May 31, 2012) requiring medical evacuation to Landstuhl Regional Medical Center (LRMC) in Germany before transfer to a participating U.S. hospital. The analysis was restricted to patients with organisms collected from wound cultures and retained in the TIDOS microbiological repository. Analysis of confirmed infections was restricted to patients with infections directly matched to a wound site. Participating U.S. hospitals were Walter Reed Army Medical Center and National Naval Medical Center in the National Capital Region (Walter Reed National Military Medical Center after September 2011) and Brooke Army Medical Center in San Antonio, Texas. The study was approved by the Institutional Review Board of the Uniformed Services University of the Health Sciences (IRB #351767).
2.2. Study Definitions
An extremity wound infection (EWI) was defined as ‘confirmed’ if the diagnosis was based on a combination of clinical (e.g., localized signs/symptoms from direct observations) and laboratory findings (e.g., microbiology and diagnostic imaging) and classified in accordance with standardized definitions of skin and soft-tissue infections (SSTI) and osteomyelitis from the National Healthcare Safety Network (Centers for Disease Control and Prevention, 2018). In absence of meeting a priori definition, infections were included if there was a clinical diagnosis (based on physician’s direct observations or clinical judgment) associated with directed antimicrobial treatment continued for ≥5 days for SSTIs and ≥21 days for osteomyelitis (infections with shorter antibiotic durations included if surgical cure performed). Infections were excluded from analysis if there was a record of an alternate diagnosis along with discontinuation of antimicrobial treatment. A ‘suspected’ infection was defined as isolation of organisms from wound cultures with associated signs and symptoms; however, it did not meet clinical diagnostic criteria of a wound infection. A third group, ‘colonization’ was defined as having organisms isolated from wounds without either a confirmed or suspected clinical infection.
Multidrug resistance was determined based on standardized definitions (Division on Healthcare Quality Promotion, 2018). Organisms resistant to ≥3 classes of antibiotics (aminoglycosides, beta-lactams, carbapenems, and fluoroquinolones) or if expressed extended-spectrum beta-lactamases or carbapenemases were classified as multidrug-resistant. Methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus were considered multidrug-resistant.
Patients in this analysis frequently sustained multiple wounds. Therefore, a patient with a confirmed EWI may have had cultures collected from additional wounds, which grew organisms. These additional wounds were classified as either suspected infection or colonization based on the presence or absence of signs and symptoms of a wound infection as described above.
2.3. Data Sources and Statistical Analysis
Information on injury patterns, severity of injury, and intensive care unit admission was obtained from the DoD Trauma Registry (Eastridge et al., 2006). Data on inpatient EWIs and microbiological findings were collected from the supplemental TIDOS infectious disease module (Tribble et al., 2011). Characteristics between patients with confirmed monomicrobial and polymicrobial EWIs were compared using Fisher’s exact test. Statistical analysis was performed with SAS® version 9.3 (SAS, Cary, NC). P<0.05 was considered statistically significant.
3.0. RESULTS
3.1. Study Population
Between 2009 and 2012, 1409 combat casualties with extremity wounds were admitted to LRMC and transferred to a participating U.S. hospital (Figure 1). A total of 323 (23% of 1409) patients had a confirmed EWI; however only 250 (77% of 323) patients met criteria of having organisms collected from wound cultures and retained in the TIDOS microbiological repository (60 patients without clinical workups and 13 patients without retained organisms excluded; Tables 1 and 2). In addition, 150 (57% of 263) patients with confirmed EWIs had organisms isolated from another wound with a suspected infection, while 42 (16%) patients had organisms recovered from separate colonized wounds.
Figure 1.
Flow diagram of extremity wound infection study population. Confirmed extremity wound infections (EWIs) were based on a combination of clinical and laboratory findings and classified in accordance with standardized definitions from the National Healthcare Safety Network. Patients with confirmed EWIs also had organisms recovered from other wounds (not confirmed to be EWI) with the results being a suspected EWI or colonization. Suspected infections were defined as wound cultures with positive growth, along with signs or symptoms of a wound infection, but did not meet clinical diagnostic criteria. Colonization was defined as organism recovery from wounds without any sign or symptom of infection.
Table 1.
Characteristics of Wounded Military Personnel with Confirmed Extremity Wound Infections, No. (%)a
| Patients with only Monomicrobial Infections (N=89) | Patients with Polymicrobial Infections (N=161) | P-value | |
|---|---|---|---|
| Operational Theater | 0.293 | ||
| Iraq | 5 (5.6) | 4 (2.5) | |
| Afghanistan | 83 (93.3) | 149 (92.5) | |
| Missing | 1 (1.1) | 8 (5.0) | |
| Limbs injured | 0.260 | ||
| 1 | 11 (12.4) | 11 (6.8) | |
| 2 | 23 (25.8) | 32 (19.9) | |
| 3 | 31 (34.8) | 67 (41.60 | |
| 4 | 24 (27.0) | 51 (31.7) | |
| Injured Limbs with Infections and Recovered Isolates | 0.008 | ||
| 1 | 73 (82.0) | 105 (65.2) | |
| 2 | 13 (14.6) | 47 (29.2) | |
| 3 | 2 (2.2) | 9 (5.6) | |
| 4 | 1 (1.1) | 0 | |
| Body region of Injury | 0.173 | ||
| Lower extremity only | 69 (77.5) | 132 (82.0) | |
| Lower and upper extremity | 7 (7.9) | 17 (10.6) | |
| Upper extremity only | 13 (14.6) | 12 (7.5) | |
| Injury severity scoreb | 0.001 | ||
| 0–9 (mild) | 2 (2.2) | 3 (1.9) | |
| 10–15 (moderate) | 4 (4.5) | 9 (5.6) | |
| 16–24 (severe) | 27 (30.3) | 17 (10.6) | |
| ≥25 (critical) | 56 (62.9) | 132 (82.0) | |
| Injury Pattern | 0.001 | ||
| Amputations | 57 (64.0) | 134 (83.2) | |
| Open fractures | 25 (28.1) | 25 (15.5) | |
| Other wounds | 7 (7.9) | 2 (1.2) | |
| Admission to LRMC ICU | 68 (76.4) | 139 (86.3) | 0.046 |
| Admission to U.S. hospital ICU | 55 (61.8) | 121 (75.2) | 0.027 |
ICU - intensive care unit; LRMC - Landstuhl Regional Medical Center
An extremity wound infection was defined as confirmed if the diagnosis was based on a combination of clinical and laboratory findings and classified in accordance with standardized definitions of skin and soft-tissue infections and osteomyelitis from the National Healthcare Safety Network (Centers for Disease Control and Prevention, 2018). In absence of meeting a priori definition, an infection was included if there was a clinical diagnosis associated with directed antimicrobial treatment.
The Injury Severity Score is an overall measure calculated for each patient based on the top three maximum Abbreviated Injury Scale anatomical region values (Linn, 1995)
Table 2.
Confirmed Monomicrobial and Polymicrobial Extremity Wound Infectionsa
| Monomicrobial Infectionsb | Polymicrobial Infectionsc | |||
|---|---|---|---|---|
| Number of Cultures (%) | Median Days from Injury to First Infection (min-max) | Number of Cultures (%) | Median Days from Injury to First Infection (min-max) | |
| Total Unique Infections | 131 | 11 (1–136) | 204 | 7 (1–120) |
| Gram-negative Bacteria | 75 (57.3) | 14 (4–47) | 175 (85.8) | 7 (1–120) |
| Acinetobacter spp. | 21 (28.0) | 8 (5–28) | 36 (20.6) | 13 (2–44) |
| Enterobacter spp. | 12 (16.0) | 17.5 (7–34) | 57 (32.6) | 4 (1–42) |
| Escherichia coli | 9 (12.0) | 14 (5–47) | 64 (36.6) | 8 (1–33) |
| Klebsiella spp. | 1 (1.3) | 10 (10–10) | 6 (3.4) | 13.5 (4–27) |
| Pseudomonas spp. | 20 (26.7) | 14.5 (4–33) | 52 (29.7) | 7 (2–120) |
| Other Gram-negatives | 12 (16.0) | 24.5 (5–36) | 59 (33.7) | 6 (1–42) |
| Gram-positive Bacteria | 30 (22.9) | 7.5 (1–136) | 146 (71.6) | 6 (1–120) |
| Coagulase-negative staphylococci | 11 (36.7) | 17 (4–69) | 17 (11.6) | 4 (1–31) |
| Enterococcus spp. | 14 (46.7) | 6 (1–18) | 108 (74.0) | 6 (2–44) |
| Staphylococcus aureus | 2 (6.7) | 76 (16–136) | 7 (4.8) | 11 (2–120) |
| Other Gram-positives | 3 (10.0) | 9 (5–27) | 55 (37.7) | 3 (1–22) |
| Mold or Yeast | 20 (15.3) | 10.5 (3–65) | 80 (39.2) | 5 (1–43) |
| Aspergillus spp. | 4 (20.0) | 8 (7–30) | 26 (32.5) | 4.5 (1–19) |
| Candida spp. | 3 (15.0) | 11 (11–65) | 15 (18.8) | 12 (2–43) |
| Order Mucorales | 4 (20.0) | 8 (5–17) | 19 (23.8) | 5 (2–15) |
| Other mold | 9 (45.0) | 11 (3–31) | 46 (57.5) | 4.5 (1–21) |
| Other yeast | 0 | NA | 2 (2.5) | 5.5 (4–7) |
| Anaerobes | 6 (4.6) | 5.5 (5–10) | 35 (17.2) | 4 (2–32) |
| Bacteroides spp. | 2 (33.3) | 5 (5–5) | 14 (40.0) | 4.5 (3–32) |
| Clostridium spp. | 2 (33.3) | 5.5 (5–6) | 16 (45.7) | 3 (2–11) |
| Other anaerobes | 2 (33.3) | 9 (8–10) | 13 (37.1) | 6 (2–18) |
An extremity wound infection was defined as confirmed if the diagnosis was based on a combination of clinical and laboratory findings and classified in accordance with standardized definitions of skin and soft-tissue infections and osteomyelitis from the National Healthcare Safety Network (Centers for Disease Control and Prevention, 2018). In absence of meeting a priori definition, an infection was included if there was a clinical diagnosis associated with directed antimicrobial treatment. Data are presented from first recorded confirmed extremity wound infection.
Blast trauma patients contributed 126 monomicrobial infections (96% of 131). All Enterococcus spp., S. aureus, E. coli, Klebsiella spp., anaerobes, mold, and Candida spp. isolates recovered from monomicrobial infections were from blast wounds.
Polymicrobial infections have more than one organism identified so the values sum to greater than the total number of infections. Blast trauma patients contributed 200 polymicrobial infections (98% of 204). All coagulase-negative staphylococci, E. coli, Klebsiella spp., Bacteroides spp., mold, and yeast isolates were collected from patients with blast trauma.
Twenty-four patients without confirmed EWIs had organisms recovered from extremity wounds with suspected infections (Table 3), while 18 patients without confirmed EWIs had organisms isolated from cultures of colonized wounds (Table 4).
Table 3.
| Suspected Wound Infection Cultures, No. | ||
|---|---|---|
| Only Monomicrobialc | Polymicrobiald | |
| Total Positive Cultures | 22 | 8 |
| Gram-Negative bacteria | ||
| Achromobacter spp. | 0 | 1 |
| Acinetobacter spp. | 3 | 4 |
| Alicaligenes denitrificans | 1 | 0 |
| Chryseobacterium spp. | 2 | 1 |
| Enterobacter spp. | 1 | 1 |
| Pseudomonas spp. | 2 | 2 |
| Gram-Positive Bacteria | ||
| Bacillus spp. | 0 | 1 |
| Coagulase-negative staphylococci | 10 | 0 |
| Enterococcus spp. | 0 | 1 |
| Mold | ||
| Aspergillus spp. | 1 | 1 |
| Order Mucorales | 0 | 1 |
| Other mold | 2 | 5 |
A suspected infection was defined as isolation of organisms from wound cultures with associated signs and symptoms; however, it did not meet clinical diagnostic criteria of a wound infection.
Total is 24 patients with suspected extremity wound infections (18 with monomicrobial cultures and 8 with polymicrobial cultures; 2 patients had more than one suspected EWI). Twenty-three (96%) patients injured were via a blast (17 with monomicrobial wounds and 8 with polymicrobial wounds). Only Alcaligenes dentrificans and one Pseudomonas spp. isolate were recovered from non-blast wounds.
Median time from injury to culture was 3 days for patients who grew mold, 15 days for Gram-positive bacteria, and 17 days for Gram-negative bacteria.
Polymicrobial cultures have more than one organism identified so the values sum to greater than the total number of cultures. Median time from injury to culture was 27 days for patients who grew bacteria alone, 6 days for bacteria plus mold, and 3 days for mold alone.
Table 4.
| Colonized Wound Cultures, No. | ||
|---|---|---|
| Only Monomicrobialc | Polymicrobiald | |
| Total Positive Cultures | 11 | 8 |
| Gram-Negative bacteria | ||
| Acinetobacter spp. | 1 | 3 |
| Enterobacter spp. | 0 | 2 |
| Escherichia coli | 1 | 2 |
| Pseudomonas spp. | 1 | 0 |
| Serratia spp. | 0 | 1 |
| Gram-Positive Bacteria | ||
| Bacillus spp. | 0 | 1 |
| Coagulase-negative staphylococci | 2 | 1 |
| Enterococcus spp. | 1 | 2 |
| Peptostreptococcus spp. | 0 | 3 |
| Mold or Yeast | ||
| Aspergillus spp. | 2 | 1 |
| Candida spp. | 1 | 0 |
| Other mold | 2 | 5 |
| Other yeast | 0 | 1 |
Colonization was defined as having organisms isolated from wounds without either a confirmed or suspected clinical infection.
Total is 18 patients with colonized wounds (11 with monomicrobial cultures and 8 with polymicrobial cultures; one patient had more than one colonized wound). Fourteen (78%) patients injured were via a blast (7 with monomicrobial wounds and 7 with polymicrobial wounds). All mold and yeast, as well as all Gram-positive bacteria from polymicrobial wounds, were recovered from blast wounds.
Median time from injury to culture was 9 days for patients who grew Gram-negative bacteria, 5 days for Gram-positive bacteria, and 3 days for mold and Candida spp.
Polymicrobial cultures have more than one organism identified so the values sum to greater than the total number of cultures. Median time from injury to culture was 6 days for bacteria only, 4.5 days for mold only, 3 days for bacteria plus mold, and 46 days for bacteria plus yeast.
3.2. Confirmed Extremity Wound Infections
Among 250 patients with confirmed EWIs, 89 (36%) had only monomicrobial infections and 161 (64%) had polymicrobial infections for a total of 335 unique infections. Blast was the mechanism of injury for 241 (96%) patients, including all 191 patients with traumatic amputations.
A higher proportion of patients with monomicrobial infections had only one limb with an infected wound and associated microbiology data compared to polymicrobial patients with infections (p=0.008; Table 1). Patients with polymicrobial infections had higher injury severity (p=0.001), more traumatic amputations (p=0.001), and a greater number were admitted to the intensive care unit at LRMC (p=0.046) and U.S. hospitals (p=0.027).
3.2.1. Monomicrobial Infections
A total of 131 unique monomicrobial infections were identified. The median time from injury to first infection diagnosis was 11 days (Table 2). Recovered isolates were primarily Gram-negative bacteria (57%) with Acinetobacter spp. and Pseudomonas spp. contributing the largest proportion (28% and 27%, respectively). In addition, 23% of isolates were Gram-positive with Enterococcus spp. accounting for the majority (47%), followed by coagulase-negative staphylococci (CNS; 37%).
Seven patients had a monomicrobial infection on two separate injured limbs (second infection diagnosed within three days of first infection), of which four patients had organisms in common between the first and second infections (two with Acinetobacter spp., one Enterococcus spp., and one Bacteriodes spp.). Among three patients with a third limb diagnosed with a monomicrobial infection (timing of third infection unspecified), one patient had an Acinetobacter spp. in common with the other infections. A single patient had monomicrobial infections in all four limbs (diagnosed same day), involving the same Acinetobacter spp. Lastly, six patients had a second infected limb diagnosed >7 days after initial infection with one patient having an organism in common with the initial EWI.
Among 90 patients with a monomicrobial infection as their first infection, clinical cultures collected after initial diagnosis were examined to assess organism persistence. A total of 105 cultures with organism growth (63 cultures with Gram-negative, 27 Gram-positive, 4 anaerobes, and 11 molds/yeast) were obtained within three days of initial infection diagnosis. Among these cultures, 34 (32%) grew MDROs, of which 32 were Gram-negative (51% of 63) and 2 were Gram-positive (7% of 27). Thirty cultures were collected 4–11 days after infection diagnosis and 14 (47%) grew the same organism (7 with Gram-negatives, 5 Gram-positives, 1 anaerobe, and 1 mold), while 6 (20%) identified a different organism (10 cultures negative). Of the 20 cultures with growth, 9 (45%) isolated MDROs (all Gram-negative). Nine cultures were collected 12–19 days post-infection diagnosis with four (44%) being concordant with the initial culture. Concordant cultures grew three Gram-negatives and one Gram-positive. One culture (11%) grew an organism that was not identified in the initial culture, while four cultures were negative. Of the five cultures that grew organisms during this period, three (60%) isolated MDROs (all Gram-negative).
3.2.2. Polymicrobial Infections
A total of 204 unique polymicrobial infections were identified with a median duration of 7 days post-injury to first infection diagnosis (Table 2). Bacteria was the only etiologic agent in 124 infections (61%), followed by bacteria plus mold (30%), bacteria plus yeast (5%), bacteria plus mold and yeast (2%), and mold alone (1%). Similar to monomicrobial infections, Gram-negative bacteria predominated with isolates recovered from 86% of infections. E. coli, Enterobacter spp., and Pseudomonas spp. accounted for the majority of isolates (37%, 33%, and 30%, respectively). Gram-positive bacteria were isolated in 72% of polymicrobial infections and Enterococcus spp. were most frequent (74%).
Forty patients had ≥2 injured limbs diagnosed with polymicrobial infections within three days of each other, of which 35 (88%) had organisms in common. Specifically, 15 patients had a polymicrobial infection on a second limb that recovered only bacterial organisms with ≥1 organism in common with the initial infection. Nineteen patients had their second infection involving a combination of bacteria, mold, and/or yeast with ≥1 concordant organism between the first and second infections. Lastly, one patient with two infected limbs that grew only fungal species had ≥1 concordant mold. Five patients had a third limb (timing unspecified) with a polymicrobial infection that included ≥1 organism in common with other infections. Seven patients had a polymicrobial EWI diagnosed in a second limb >7 days after initial EWI diagnosis with two patients having ≥1 concordant organism with the initial infection.
Among 157 patients with a polymicrobial infection as their first infection diagnosis, 197 cultures with organism growth were collected within three days of diagnosis. Enterococcus spp. was the most frequent organism (108 cultures; 55%), while E. coli was the most common Gram-negative organism (64 cultures; 32%). Multidrug resistance was identified in 87 cultures (44%), of which 68 and 19 cultures grew multidrug-resistant Gram-negative and Gram-positive organisms, respectively. During the first week after infection diagnosis (4–11 days post-diagnosis), 81 subsequent cultures were collected and 65 (80%) grew a combination of organisms concordant with initial cultures, as well as those that were not previously recovered (16 cultures negative). Organisms that were concordant were predominantly E. coli, Enterococcus spp., Pseudomonas spp., Acinetobacter spp., and molds. Approximately half of cultures with growth (33 cultures) identified MDROs (28 with Gram-negative growth, 3 with Gram-positive, and 2 with Mycobacterium spp.). On days 12–19 post-infection diagnosis, 25 cultures were collected and 15 (60%) identified organisms concordant with the initial infection along with new organisms (10 cultures negative). Concordant organisms were primarily Enterococcus spp., Acinetobacter spp., and Pseudomonas spp. Of the 15 cultures that grew organisms during this period, 5 (33%) isolated MDROs (all Gram-negative).
3.2.3. Suspected Infections Among Patients with Confirmed EWIs
Among 263 patients with confirmed EWIs and clinical workups, 150 had organisms recovered from 182 other wounds with suspected infections, resulting in 68 patients with a suspected monomicrobial infection and 114 with a suspected polymicrobial infection (32 patients had both monomicrobial and polymicrobial infections). A total of 142 subjects had organisms recovered from both confirmed EWIs and separate wounds with suspected infections, of which 136 (96%) had ≥1 organism in common at genus-level. Seventy-one (50%) subjects had the same genus-level microbiological profile between confirmed EWIs and other wounds with suspected infections (26 subjects had only one organism recovered).
A total of 89 monomicrobial cultures were collected from patients with suspected monomicrobial infections from other wounds, which primarily identified Gram-negative bacteria (40 cultures; 45%) with Acinetobacter spp. contributing the largest proportion (14 cultures; 35%). Among Gram-positive growth (27 cultures; 30%), Enterococcus spp. and CNS were recovered in 9 cultures (each 33%). Less cultures grew mold and yeast (16 cultures; 18%) and anaerobes (6 cultures; 7%). Isolates were identified a median of 11 days post-injury for Gram-negative organisms, 11 days for mold and yeast, 15 days for Gram-positives, and 33 days for anaerobes.
Regarding suspected polymicrobial infections among patients with confirmed EWIs, 114 cultures were collected, of which 63 (55%) cultures only recovered bacteria, while 32 (28%) were a combination of bacteria and mold, 9 (8%) had bacteria with both yeast and mold, 5 (4%) had bacteria plus yeast, and 5 (4%) only had mold. Among Gram-negative bacteria, E. coli, Pseudomonas spp., and Enterobacter spp. were predominant, while Enterococcus spp. was the largest contributor of Gram-positive bacteria. The majority of molds were Aspergillus spp., mold from the order Mucorales, and other molds, while it was primarily Candida spp. for yeasts. Lastly, Clostridium spp. and Bacteroides spp. accounted for the majority of anaerobes. Cultures with only bacterial growth were identified a median of 11 days post-injury, while it was 3.5 days for bacteria plus mold, 5 days for mold only, 5 days for bacteria plus mold and yeast, and 16 days for bacteria plus yeast.
3.2.4. Colonization Among Patients with Confirmed EWIs
Forty-two patients with confirmed EWIs had organisms recovered from non-infected wounds (colonization), of which 27 and 21 had monomicrobial and polymicrobial colonized wounds, respectively (6 patients had >1 colonized wound). A total of 31 subjects had organisms recovered from both confirmed EWIs and separate colonized wounds, of which 16 (52%) had ≥1 organism in common at genus-level. Only one subject had the same microbiological profile between the confirmed EWI and colonized wound.
A total of 30 cultures were collected from wounds with monomicrobial colonization and mold and yeast were the largest contributor with 19 cultures (63%), followed by Gram-positive bacteria (5 cultures, 17%), Gram-negative bacteria (3 cultures, 10%), and anaerobes (3 cultures, 10%). Among cultures with mold and yeast, Aspergillus spp., mold from the order Mucorales, and other molds were predominant. Gram-positive bacteria included Enterococcus spp., Bacillus spp., and CNS, while Gram-negative bacteria were Enterobacter spp. and Morganella spp. Anaerobes recovered from cultures were Bacteroides spp. and Propionibacterium spp. Isolates were collected a median of four days post-injury for Gram-negative bacteria and three days for Gram-positive bacteria, anaerobes, mold, and yeast.
Twenty-one cultures collected from colonized wounds were polymicrobial, of which the majority were a combination of bacteria and mold (12 cultures, 57%) followed by only mold (6 cultures, 29%) and only bacteria (3 cultures, 14%). Mold and Candida spp. accounted for the largest proportion (18 cultures, 86%) with Aspergillus spp. and mold other than from the order Mucorales being the predominant isolates. Bacillus spp. and Enterococcus spp. were largely recovered from cultures that grew Gram-positive bacteria, while Enterobacter spp. was the greatest contributor of Gram-negative bacteria. Only one culture grew an anaerobe (Clostridium spp.). Isolates from polymicrobial cultures were identified a median of 2 to 3 days post-injury.
4.0. DISCUSSION
Extremity wounds are the most frequent injury pattern associated with combat-related trauma and often result in infectious complications. Our study found wound infections are predominantly the result of Gram-negative bacteria (57% and 86% of monomicrobial and polymicrobial infections) with E. coli, Enterobacter spp., Pseudomonas spp., and Acinetobacter spp. frequently isolated. Gram-positive bacteria were recovered from 72% of polymicrobial confirmed EWIs with Enterococcus spp. contributing the majority. While mold/yeast and anaerobes were recovered at a lower proportion (40% and 17% of polymicrobial confirmed EWIs, respectively), they cannot be discounted and warrant consideration. Over half of wounds with infections are polymicrobial with 38% growing a combination of bacteria plus mold and/or yeast.
Although they are less frequent, infections involving mold, Gram-positive bacteria, and anaerobes have been observed among combat casualties. In particular, 6.8% of combat casualties injured between 2009 and 2011 developed an invasive fungal wound infection (IFI). Nonetheless, presence of mold from wound cultures alone is not sufficient to diagnose IFIs. For a wound with mold culture growth to be classified as a possible IFI requires recurrent wound necrosis following ≥2 surgical debridements. Wounds meeting these criteria should also have tissue specimens sent for histopathological analysis to diagnose IFI with a higher degree of certainty (Weintrob et al., 2015). Without the hallmark of recurrent wound necrosis, wounds that grow mold can be considered colonized and do not require antifungal therapy (Rodriquez et al., 2014).
Obligate anaerobes and Candida spp. isolates recovered from combat casualties have also been examined. In an analysis of anaerobes (primarily Bacteroides spp.) collected from wound cultures from 59 combat casualties, 85% of isolates were recovered from infected wounds. Although 6.8% of patients with anaerobe recovery died, the pathogenic role of anaerobes is uncertain and their occurrence may be a marker of disease severity (White et al., 2016). Candida spp. were also isolated from wounds with confirmed EWIs in our analysis, as well as suspected infections and colonization. In a prior study, 131 Candida spp. isolates recovered from combat casualties were evaluated and included 37 (28%) isolates collected from wound cultures, of which 86% were associated with infections. As there was no significant association between Candida isolation and mortality, it is presumably that Candida spp. isolation resulted from frequent culture collection due to high injury severity and was not an indication of pathogenicity (Blyth et al., 2014).
The large proportion of polymicrobial wounds in our study (64% of patients with confirmed EWIs) is consistent with prior reports of combat extremity trauma (Petersen et al., 2007; Wallum et al., 2015). Specifically, an assessment of 14 mangled lower extremities that underwent amputation found that wounds were polymicrobial and frequently grew both Gram-negatives and Gram-positives with a smaller proportion identifying mold and/or yeast. The Gram-negative bacteria were generally Pseudomonas spp., while Enterococcus spp., represented the majority of Gram-positive isolates (Wallum et al., 2015). An additional study found that Enterococcus spp. were the highest bacterial contributor to polymicrobial SSTI and fungal-infected wounds from blast trauma (Warkentien et al., 2015). Polymicrobial infections was also frequently observed in an analysis of patients with enterococcal wound infections (Heitkamp et al., 2018). Although E. coli and Enterobacter spp. were the primary Gram-negative organisms identified from polymicrobial confirmed EWIs in our study, Pseudomonas spp. still contributed a high proportion. Moreover, over 70% of cultures collected from polymicrobial wounds grew Gram-positive bacteria, which were predominantly Enterococcus spp.
The microbiological profile of confirmed and suspected EWIs from the same patient were similar (96% with ≥1 organism in common) with Acinetobacter spp., E. coli, Pseudomonas spp., CNS, and Enterococcus spp. as the predominant organisms. While patients in the suspected EWI and colonization populations (N=24 and 18, respectively) did not have anaerobe growth, anaerobes were isolated from wounds of confirmed EWI patients (both confirmed infections and wounds with suspected infections and colonization). Unlike confirmed and suspected EWIs, cultures collected from colonized wounds were largely monomicrobial (58%) and primarily identified molds; however, bacteria (Enterococcus spp. and Acinetobacter spp.) were still recovered.
Our findings are comparable with prior investigations of wound microbiology. In an analysis of civilian trauma patients injured in an earthquake in China, wound infection cultures primarily recovered E. coli, Enterococcus spp., Acinetobacter baumannii, and S. aureus (Zhang et al., 2012), which was similar to the bacterial distribution from infected wounds in our study. Regarding combat-related wounds, an examination of extremity wounds (predominantly lower extremities) found that A. baumannii accounted for 63% of identified isolates followed by Enterococcus faecium (12%) and E. coli (7%). Among cultures collected during the first week post-injury, 7.7% were polymicrobial compared to 2.9% and 2.0% in the second and third weeks. A. baumannii remained the largest bacterial contributor across three weeks, while E. faecium increased during the second week and decreased in the third. In contrast, E. coli decreased in the second week, but rose in the third (Sheppard et al., 2010). Our analysis found a high proportion of Acinetobacter spp. recovered from patients with confirmed and suspected EWIs (both monomicrobial and polymicrobial cultures). Furthermore, 22% and 20% of cultures with E. coli and Enterococcus spp. identified in the initial polymicrobial EWI had the same organisms recovered a week later.
Our study does have limitations that should be considered. Our population consisted of severely injured combat casualties (76% of patients had traumatic amputations); therefore, the wound microbiology data may not be generalizable to all wounded personnel. Furthermore, we do not have a way of definitively determining which organisms isolated in polymicrobial wounds were the true pathogen.
4.1. Conclusions
Gram-negative bacteria are predominant in both monomicrobial and polymicrobial confirmed EWIs, while Gram-positive bacteria also substantially contributed to polymicrobial EWIs. Combat casualties with confirmed EWIs often had additional colonized wounds or wounds with suspected infections. The majority of suspected and colonized wounds shared ≥1 organism in common with the confirmed EWI on the same subject. Overall, the results of our study confirm the complexity of combat-related wounds with cultures frequently growing multiple bacteria, as well as anaerobes, mold, and yeast. Describing the microbiome of these combat extremity wounds leads to a better understanding of the complexity of and infection burden, which will result in improved wound management and infection outcomes in future conflicts by applying appropriate wound care and antimicrobial therapies. Furthermore, infection control is an important component of patient care to prevent nosocomial transmission of pathogens. Knowledge of the wound microbiome will lead to improvements in infection control strategies, which may result in less infectious complications and the prevention of nosocomial transmission. Future investigations will focus on outcomes related to specific microbiology, particularly Enterococcus spp., and risk factors for wound infection.
Acknowledgements:
We are indebted to the Infectious Disease Clinical Research Program Trauma Infectious Disease Outcomes Study team of clinical coordinators, microbiology technicians, data managers, clinical site managers, and administrative support personnel for their tireless hours to ensure the success of this project.
Financial Support: Support for this work (IDCRP-024) was provided by the Infectious Disease Clinical Research Program (IDCRP), a Department of Defense program executed through the Uniformed Services University of the Health Sciences, Department of Preventive Medicine and Biostatistics. This project has been funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health [under Inter-Agency Agreement Y1-AI-5072], the Department of the Navy under the Wounded, Ill, and Injured Program [grant number HU001-10-0014], Defense Medical Research and Development Program, and the Military Infectious Disease Research Program [grant number HU0001-15-2-0045].
Footnotes
Presented in part: 2016 Infectious Disease Society of America ID Week, 26–30 October 2016, New Orleans, LA.
Conflicts of Interest: None
Disclaimer: The view(s) expressed are those of the authors and do not reflect the official views of the Uniformed Services University of the Health Sciences, Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., the National Institutes of Health or the Department of Health and Human Services, Brooke Army Medical Center, Walter Reed National Military Medical Center, Landstuhl Regional Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of Defense, or the Departments of the Army, Navy or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.
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