Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients.

Recent reclassification of the Klebsiella genus to include Klebsiella variicola, and its association with bacteremia and mortality, has raised concerns. We examined Klebsiella spp. infections among battlefield trauma patients, including occurrence of invasive K. variicola disease. Klebsiella isolates collected from 51 wounded military personnel (2009-2014) through the Trauma Infectious Disease Outcomes Study were examined using polymerase chain reaction (PCR) and pulsed-field gel electrophoresis. K. variicola isolates were evaluated for hypermucoviscosity phenotype by the string test. Patients were severely injured, largely from blast injuries, and all received antibiotics prior to Klebsiella isolation. Multidrug-resistant Klebsiella isolates were identified in 23 (45%) patients; however, there were no significant differences when patients with and without multidrug-resistant Klebsiella were compared. A total of 237 isolates initially identified as K. pneumoniae were analyzed, with 141 clinical isolates associated with infections (remaining were colonizing isolates collected through surveillance groin swabs). Using PCR sequencing, 221 (93%) isolates were confirmed as K. pneumoniae, 10 (4%) were K. variicola, and 6 (3%) were K. quasipneumoniae. Five K. variicola isolates were associated with infections. Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p = 0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01). No K. variicola isolates demonstrated the hypermucoviscosity phenotype. Although K. variicola isolates were frequently isolated from bloodstream infections, they were less likely to be multidrug-resistant. Further work is needed to facilitate diagnosis of K. variicola and clarify its clinical significance in larger prospective studies.

Introduction

Klebsiella pneumoniae is recognized as a frequent cause of healthcare-associated infections, including bloodstream infections (BSI), urinary tract infections, ventilator-associated pneumonia, and surgical site infections [1]. Among military personnel with battlefield injuries sustained in Iraq and Afghanistan, K. pneumoniae was one of the most common colonizing Gram-negative bacilli identified from groin surveillance cultures collected at hospital admission, with approximately 22% of isolates being classified as multidrug-resistant (MDR) [2,3]. Furthermore, K. pneumoniae was the third most frequently identified isolate from wounded warriors with MDR Gram-negative bacilli infections [4].

Re-classification of the Klebsiella genus and description of new Klebsiella species has raised questions regarding species specific virulence [5-9]. Certain themes have emerged from early research done after this re-classification. Most notably, there is an association between Klebsiella variicola and healthcare-associated infections with potential for worsened outcomes and greater invasive disease (e.g., bacteremia) when compared to K. pneumoniae [5-10]. However, most modern clinical laboratories misidentify K. variicola as K. pneumoniae and only recently has polymerase chain reaction (PCR) and mass spectrometry techniques for identification been described [11-13]. Thus, epidemiology, clinical manifestations, and virulence patterns of K. variicola remain controversial.

With the importance of K. variicola as an emerging pathogen, we evaluated Klebsiella infections in patients who suffered battlefield-related trauma [8]. Specifically, we examined the epidemiology and resistance patterns of Klebsiella spp. infections, identified prior misclassifications of Klebsiella species, and assessed the prevalence and incidence of invasive K. variicola disease in this population.

Materials and methods

Study population and definitions

Data were collected through the Trauma Infectious Disease Outcomes Study (TIDOS), which is a retrospective observational study of infectious complications among military personnel wounded in Iraq or Afghanistan (2009–2014) [14,15]. All patients in the TIDOS population were ≥18 years of age active-duty personnel or Department of Defense (DoD) beneficiaries who initially received care in the combat theater, followed by medical evacuation to Landstuhl Regional Medical Center (LRMC) in Germany with ultimate transfer to participating military hospitals in the United States. The participating U.S. military hospitals were Brooke Army Medical Center in San Antonio, TX, and Walter Reed National Military Medical Center in the National Capital Region (National Naval Medical Center and Walter Reed Army Medical Center prior to September 2011) [14].

The Institutional Review Board (IRB) of the Uniformed Services University (Bethesda, MD) approved this study. Data were collected from subjects that provided authorization for the collection and analysis of their data through informed consent and HIPAA authorization processes, or through an IRB-approved waiver of consent for use of de-identified data not obtained through interaction or intervention with human subjects.

Inclusion in this analysis required isolation of a Klebsiella spp. isolate associated with clinical diagnosis of infection. Demographics, trauma characteristics, and information on casualty care were collected from the DoD Trauma Registry (DoDTR) [16] and infection-related information (e.g., infection syndromes, microbiology, and antibiotic treatment) was obtained from the TIDOS Infectious Disease module of the DoDTR. Infectious disease events were identified using a combination of clinical and laboratory findings and classified using National Healthcare Safety Network definitions as previously described [14,17]. Colonization was defined as recovery of isolates from groin swabs obtained as part of targeted infection control surveillance at hospital admission for their deployment-related injury. All other isolates were defined as infecting isolates as they were recovered during workups for clinical infection. Multidrug resistance was defined using Centers for Disease Control and Prevention criteria as either resistance to ≥3 classes of aminoglycosides, β-lactams, carbapenems, and/or fluoroquinolones or production of an extended spectrum beta-lactamase (ESBL) or carbapenamase [18].

Klebsiella spp. isolate analysis

Initial identification of organisms was performed by participating hospitals’ clinical microbiology laboratories. All hospital labs used either BD Automated Microbiology System (BD Diagnostics, Sparks, MD) or Vitek 2 (bioMérieux Inc., Hazelwood, MO). These two platforms are only able to identify K. pneumoniae and not differentiate this identification from K. variicola, or other members of the Klebsiella complex [9]. Isolates were stored at -80⁰C in a central TIDOS specimen repository.

All initial and serial infecting isolates identified as K. pneumoniae and stored in the TIDOS specimen repository were included for analysis. Serial isolation was defined as an isolate collected ≥7 days from a prior isolate. All colonizing isolates linked with infecting isolates (defined as isolation from groin admission swab prior to infection) were included. A convenience sample of the remaining colonizing isolates (50 MDR and 50 non-MDR colonizing K. pneumoniae archived isolates) were chosen randomly from the repository.

All isolates underwent passage on 5% sheep blood agar twice prior to confirmatory identification and antimicrobial susceptibility testing utilizing BD Phoenix Gram-negative panel (NMIC/ID-304) and BD Automated Microbiology System (BD Diagnostics, Sparks, MD)–this technique replicates typical clinical laboratory procedures and is only able to identify K. pneumoniae. Breakpoints were determined using Clinical Laboratory Standards Instituted guidelines (M100, 28th edition, 2018). Antimicrobials tested included cefazolin, ceftriaxone, cefepime, levofloxacin, amoxicillin-clavulanate, piperacillin-tazobactam, aztreonam, meropenem, ertapenem, amikacin, and trimethoprim/sulfamethoxazole.

All isolates underwent DNA extraction using QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany) and subsequently pulsed-field gel electrophoresis (PFGE) analysis to assess for clonality. Any uninterpretable gel patterns on PFGE were repeated. In order to identify K. pneumoniae, K. quasipneumoniae, and K. variicola, all extracted DNA samples underwent PCR using the method and oligonucleotide primer sequences described by Garza-Ramos and colleagues [12,19]. Of note, this work was completed prior to the extensive molecular epidemiologic work resulting in the further expansion of the Klebsiella pneumoniae complex [8,9]. Due to early case reports of K. variicola being linked with hypermucoviscosity phenotypes, string tests were performed on all K. variicola isolates. String test length for positivity was defined as stranding from one colony >5 millimeters from the agar surface. We did not perform the string test on K. pneumoniae or K. quasipneumoniae isolates as our focus was describing any association between the hypermucoviscous phenotype and K. variicola.

Statistical analysis

All patients with isolation of Klebsiella spp. were initially analyzed as a group. Patients who were subsequently identified to have K. variicola isolation by PCR assay were evaluated in a secondary analysis to examine clinical predictors of isolation and phenotypic behaviors. Univariate analysis by Χ2 and Fisher’s Exact Test was performed for categorical variables where appropriate. Continuous variables were analyzed using Mann-Whitney U. Statistical analysis was performed using IBM SPSS Statistics 22 (Version 22 IBM, NY, 2013). A p value of <0.05 was considered significant. Data availability: All relevant data are provided within the paper and its supporting documentation.

Results

Study population

Among 2,699 TIDOS patients, 51 patients with infecting Klebsiella isolates met inclusion criteria for the analysis. All patients were young men with a median age of 23 years who were severely injured with 82% sustaining blast trauma, largely from improvised explosive devices (Table 1). All 51 patients received antibiotics prior to recovery of infecting Klebsiella spp. isolates. The most common antibiotics administered prior to recovery of an infecting isolate were tetracyclines (N = 46, 90%), first generation cephalosporins (N = 45, 88%), and vancomycin (N = 39, 76%). Duration of hospitalization was a median of 49 days. Overall, four (8%) patients died.

Table 1

Characteristics of patients with Klebsiella species infections.

Characteristic or outcome, No. (%)	Patients with Klebsiella spp. isolates N = 51
Age, years, median (IQR)	23 (21–28)
Male sex	51 (100)
Injury severity score, median (IQR)	38 (15–45)
Injury Mechanism
Blast injury	42 (82)
Improvised explosive device	37 (72)
Gunshot wound	6 (12)
Initial facility geographic locationa
Southern Afghanistan:	34 (67)
Bastion	13 (25)
Role 2a	1 (2)
Kandahar	18 (35)
Role 2b	1 (2)
Role 2c	1 (2)
Eastern/Northeastern Afghanistan:	10 (20)
Role 2d	1 (2)
Role 2e	1 (2)
Role 2f	3 (6)
Role 2g	1 (2)
Role 2h	1 (2)
Role 2i	2 (4)
Role 2j	1 (2)
Central Afghanistan:	2 (4)
Bagram	1 (2)
Role 2k	1 (2)
Iraq	1 (2)
Landstuhl Regional Medical Center (Germany)	2 (4)
Other	2 (4)
U.S. military hospital
Brooke Army Medical Center	24 (47)
National Capital Region	26 (51)
Use of Mechanical ventilation	42 (82)
Antibiotic exposure prior to isolation of Klebsiella spp.	51 (100)
Days between injury and 1st infecting isolate, median (IQR)	15 (8–33)
Total length of hospital stay, median days (IQR)	49 (28–70)
Death	4 (8)

IQR–interquartile range.

a Role 2 facilities are within the operational theater with a tent or structure-based operating room and limited personnel (mobile forward surgical teams for initial and resuscitative care are included). Bastion, Kandahar, and Bagram are Role 3 facilities, which are combat support hospitals within the operational theater.

Sources of the initial 51 infecting Klebsiella isolates were respiratory (N = 16, 31%), wound (N = 13, 26%), blood (N = 10, 20%), urine (N = 5, 10%), intra-abdominal (N = 4, 8%), and other (N = 3, 6%). There were a median of 23 (interquartile range [IQR]: 22–55) days between isolation of initial infecting isolate and death for the four patients who died.

Twenty-three patients (45%) had initial Klebsiella spp. infections that were MDR. When compared to patients with initial non-MDR Klebsiella spp. infections (N = 28, 55%), there was no significant difference in age (median of 22 years [IQR: 21–29] with MDR infections versus 22 years [IQR: 21–26] for non-MDR infections, p = 0.42), or duration between injury and isolation of first infecting isolate (median of 10 [IQR: 7–27] days versus 19 [IQR: 9–37] days, p = 0.25). In addition, injury severity was similar between the two groups with patients who had MDR Klebsiella infections having a median injury severity score (ISS) of 38 (IQR: 32–46) compared to a median of 35 (IQR: 28–45; p = 0.65) for patients with non-MDR Klebsiella infections.

Sixteen (31% of 51) patients had serial isolation of Klebsiella spp. with sources being respiratory (N = 7, 43%), wound (N = 4, 25%), blood (N = 3, 19%), and urine (N = 2, 13%). Age, ISS, and days between injury and first infecting isolate of patients who had serial isolation of any Klebsiella spp. and those without (N = 35, 69%) were not statistically significant; however, patients with serial Klebsiella isolation trended towards higher mortality (19%) compared with patients who only had initial isolation (3%, p = 0.07; Table 2).

Table 2

Patients with serial isolation of Klebsiella spp. versus those with initial isolation only.

Characteristic, median (IQR)	Patients with single isolates (N = 35)	Patients with serial isolation (N = 16)	P-value
Age, years	22 (21–28)	22 (21–28)	0.23
Injury Severity Score	37 (30–45)	28 (30–45	0.42
Days between injury and first infecting isolate	16 (8–34)	15 (8–33)	0.81
Length of hospital stay, days	41 (29–59)	62 (26–80)	0.41
Death, No (%)	1 (3)	3 (19)	0.07

IQR–interquartile range.

Pulsed-field gel electrophoresis patterns

There were 12 unique PFGE patterns/types (PFTs) that were each identified in >1 patients with PFT 78 recovered from ten unique patients and PFT 80 from three unique patients. The remaining 10 PFTs were identified in two patients each. Seven (58% of 12) PFTs were associated with infections; however, none of the strains were isolated from patients who were treated at the same initial facilities. There were five (42%) unique PFTs identified among colonizing isolates recovered from patients who were evacuated from the same initial facility (Bastion, Afghanistan). Only one of these PFTs (PFT 78; identified from five colonized-patients) was identical to the PFT of an infecting isolate from a single patient (Table 3). The infecting isolate was recovered from a wound infection eight days after the first colonizing isolate was collected from the other patients. Isolation of this strain with PFT 78 from patients evacuated from Bastion ended four months after the first isolate was recovered. All infecting and colonizing isolates corresponding to PFT 78 were MDR.

Table 3

Outbreak analysis of the single pulsed-field gel electrophoresis type (PFT) 78.

Patient	PFT	Initial Location	Date of isolation	Source of initial clonal isolates
				Wound (infecting)	Groin (colonizing)
I	78	Bastion	16 August 2010	0	1
J	78	Bastion	16 August 2010	0	1
K	78	Bastion	21 August 2010	0	1
L	78	Bastion	23 August 2010	0	1
M	78	Bastion	24 August 2010	1	0
N	78	Bastion	3 December 2010	0	1

Isolate analysis

Two hundred and thirty-seven isolates from 121 patients included in the study were initially identified as K. pneumoniae, of which 141 were infecting isolates and 96 were colonizing isolates (four colonizing isolates initially chosen were excluded after discovering they were identified incorrectly as Klebsiella species). After undergoing PCR, 221 (93% [95% confidence interval [CI]: 90–96]) isolates were verified as K. pneumoniae (from 108 patients; 134 infecting isolates, 87 colonizing isolates from groin swabs), 10 (4% [95% CI: 1.5–6.5]) were identified as K. variicola (from 8 patients; 5 infecting isolates, 5 colonizing isolates), and 6 (3% [95% CI: 0.8–5.2]) as K. quasipneumoniae (from 5 patients; 2 infecting isolates, 4 colonizing isolates). The years of K. pneumoniae collection were 28 (13% of 221) isolates in 2009, 95 (43%) in 2010, 30 (14%) in 2011, 51 (23%) in 2012, 13 (6%) in 2013, and 4 (2%) in 2014. There were no K. variicola isolates collected in 2009, while three were collected in 2010, four were collected in 2011, and one isolate was collected per year from 2012–2014. Four of the K. quasipneumoniae isolates were collected in 2011, one isolate in 2013, and one isolate in 2014.

Sources of the 134 K. pneumoniae infecting isolates were wound (N = 57, 42%), respiratory (N = 29, 22%), blood (N = 24, 18%), intraabdominal (N = 4, 3%), and other (N = 20, 15%). Of the 68 initial infecting Klebsiella spp. isolates, 43 were MDR (63%). Similarly, 11 (65%) of 17 colonizing isolates obtained prior to infecting isolates were MDR. Antimicrobial susceptibility patterns demonstrated substantial resistance to cephalosporins, fluoroquinolones, and piperacillin/tazobactam (Table 4). Five infecting isolates recovered after non-MDR colonizing isolates from the same patient demonstrated resistance to new antimicrobials: two developed resistance to cefazolin at 10 and 17 days after colonization, one to piperacillin/tazobactam at 19 days, one to ertapenem at 7 days, and one to gentamicin at 200 days after colonization.

Table 4

Antimicrobial susceptibilities of Klebsiella variicola compared with Klebsiella pneumoniae isolates, No. (%).

Antimicrobial	K. pneumoniae susceptibility (N = 221)	K. variicola susceptibility (N = 10)	P-value
Cefazolin	50 (22)	8 (80)	<0.01
Ceftriaxone	73 (33)	10 (100)	<0.01
Cefepime	81 (37)	10 (100)	<0.01
Levofloxacin	135 (61)	9 (90)	0.09
Piperacillin-tazobactam	99 (44)	10 (100)	<0.01
Meropenem	213 (96)	10 (100)	1.00
Amikacin	200 (90)	10 (100)	0.31

Klebsiella variicola

There were 10 (4% of 237 Klebsiella isolates) isolates of K. variicola identified from eight patients (7%). Five isolates were colonizers from groin swabs, while the five isolates associated with infections were from blood (N = 4), and intraabdominal specimens (N = 1). Among 51 patients with infections, 48 (94%) had infections attributed to K. pneumoniae and 3 (6%) had K. variicola infections. The three patients with K. variicola infections had a median age of 25 years (IQR: 21–46), median ISS of 38 (IQR: 30–45), and all were injured by improvised explosive devices. There were no statistically significant differences between these characteristics and those of patients with K. pneumoniae infections.

Four of the five K. variicola isolates were collected from blood specimens. In comparison, 24 (18%) of 134 K. pneumoniae infecting isolates were from blood specimens (p = 0.04). No infecting K. variicola isolates were MDR compared to 99 (74%) of infecting K. pneumoniae (p<0.01). All K. variicola isolates were string test negative.

The PFGE analysis demonstrated eight genetically unique K. variicola isolates (Fig 1). The three infecting K. variicola isolates identical by PFGE were isolated from one patient’s blood cultures on days 28 and 29 post-injury. Three patients with unrelated K. variicola isolates were treated in the same Kandahar facility (Afghanistan) over a 12-month period. The remaining two patients with K. variicola were treated in the same facility in Bastion province (Southern Afghanistan) 15 months apart, but were unique strains by PFGE.

Fig 1

Pulsed-field gel electrophoresis analysis of 10 K. variicola isolates collected from wounded military personnel.

Discussion

This study emphasizes the challenging resistance patterns of K. pneumoniae infections complicating battlefield trauma, even on initial isolation. It is also the first analysis of a broad archive of K. pneumoniae samples to systematically evaluate for K. variicola misidentification. Notably, while K. variicola infections were more likely to be associated with bacteremia, they were less likely to be MDR.

Gram-negative resistance is thought to develop in response to antimicrobial pressures and our patients are no exception as all had prolonged hospital stays and antimicrobial exposure prior to isolation of MDR Klebsiella infections. While there were no statistically significant differences between those with initial MDR compared with non-MDR infections, this is likely due to small numbers of patients. Similar to evidence in this study, evaluation of trauma patients either in developing countries or countries experiencing conflicts have demonstrated high rates of MDR Gram-negative infections [20-22]. For example, civilian trauma patients from the ongoing conflict in Syria were noted to have rates of MDR K. pneumoniae infections >80% [23]. Furthermore, nearly 74% of infecting K. pneumoniae isolates from a group of South African trauma patients were MDR [20]. These studies, along with others, have not only demonstrated high rates of MDR Gram-negative trauma-associated infections, but also confirmed risk factors for MDR infections to include older age, higher ISS, and prolonged intensive care unit length of stay [24-26]. Our data, which revealed a median of 15 days between injury and Klebsiella spp. isolation among these severely injured patients, also emphasizes the nosocomial nature of this pathogen. Adherence to infection control practices and antimicrobial stewardship practices are vital to mitigating the challenge presented by MDR organisms [27-29].

Three important points can be highlighted regarding K. variicola. One, this is the first study to systematically evaluate and comprehensively assess rates of misidentification of K. variicola in a unique trauma patient population. The overall incidence of K. variicola isolates in our battlefield wounded group (4%) is on the lower end of the range that has been described in the literature. In one study, Maatallah and colleagues [7] reported on K. variicola BSIs where they noted that 24% of K. pneumoniae isolates over a three-year period (2007–2009) were misidentified initially and later correctly identified as K. variicola. An outbreak of BSI in neonates in a Bangladesh neonatal intensive care unit also described a high rate of K. variicola (38% among 36 bacteremia patients) [6]. Our data identified a relatively small incidence of K. variicola (4% [95% CI: 1.5–6.5]), which is likely related to our use of a broader trauma population, rather than focusing on a specific source of infection, like bloodstream isolates. The findings of our study are similar to those described by Rodriguez-Medina and colleagues [8] who examined 1,060 K. pneumoniae clinical isolates from a group of hospitals in Mexico and reported a K. variicola prevalence of 2.1%. Furthermore, Long and colleagues [30] while examining misidentification in ESBL-producing K. pneumoniae, identified nearly 2% as K. variicola. The overall proportion of misidentified K. variicola bloodstream isolates (4 isolates misidentified; 14% of 28) in our study is comparable to these prior published reports. This could suggest that beyond BSIs, misidentification of K. variicola is uncommon; however, further analysis is warranted.

The second notable point is that four of the five infecting K. variicola isolates were identified from blood. Even though these are small numbers, coupled with what has been published previously, it does raises important questions. Is there some characteristic of K. variicola that leads to an increased propensity to invade the bloodstream? And does this, or some other characteristic portend worse patient outcomes? When Maatallah and colleagues published their work on K. variicola BSIs in 2014, the association with K. variicola BSI and mortality was alarming (30-day mortality approached 30% compared to 13.5% with K. pneumoniae) [7]. Concerns continued to be raised by a study reporting 54% mortality amongst patients who were infected with K. variicola in a Bangladesh neonatal intensive care unit [6]. Description of the first hypermucoviscous K. variicola organism in 2015 raised further questions that hypermucosviscosity might be an underlying mechanism to explain its invasive behavior [11]. While our findings did show an association between K. variicola and BSIs, there was no increase in mortality in these patients and none showed the hypermucoviscosity phenotype. Specifically, no patients with K. variicola infections in our analysis died, in contrast to worrisome mortality rates reported previously. It is important to note that the lack of the hypermucoviscous phenotype does not necessarily exclude a particular isolate from the hypervirulent phenotype [31].

The third point is that K. variicola isolates in this group were significantly more susceptible to first-line antimicrobials than K. pneumoniae isolates (Table 4). Data on susceptibility patterns for K. variicola are sparse due to the limited number of studies published; however, greater susceptibility does seem to be a common emerging theme. In particular, Maatalah and colleagues reported no MDR patterns in 34 K. variicola isolates from their repository analysis [7]. Moreover, assessment of K. pneumoniae (6 isolates) and K. variicola (2 isolates) isolated from a single patient demonstrated that while K. pneumoniae was imipenem-resistant, K. variicola was susceptible [32]. Our findings provide further evidence that while K. variicola may be more likely to be invasive, it is less likely to be MDR.

Our analysis is limited by the small numbers of Klebsiella infections and isolates, which may have impacted the ability to detect statistical differences between patients with MDR and non-MDR infection. As suggested by the variation in number of isolates over the years of the study, some of this limitation is likely related to the slowing rate of combat evacuations as the conflicts de-escalated and with the end of combat operations in Afghanistan by the end of 2014. Nevertheless, the findings provide useful information to help with rank ordering likelihood of this specific infection in trauma patients with these characteristics. Our second limitation lies in the PFGE analysis, which necessarily provides a broader view of relatedness among isolated organisms. However in this case, PFGE provides an important first pass analysis for clonality among isolates and argues that similarities between isolates that were associated with the Bastion group of patients, the overlapping location, temporal association of their isolation, and historical challenges with infection control in the deployed environment argues in favor of an outbreak [27,33]. Lastly, the analysis is limited by the lack of granularity in the MDR analysis–for example, further work would be helpful to include mechanisms of resistance such as de-repressed AmpC. The role K. variicola plays in healthcare-associated infections and outbreaks has been previously suggested [6,7] and further study of the Klebsiella genus over the last four years [8,30] has rapidly expanded the understanding of how the species variicola fits within the broader complex.

Overall, our data support findings from previous studies from infections complicating combat-trauma: these patients were severely injured, had prolonged hospital stays with exposure to antibiotics, and K. pneumoniae infections were marked by a high rate of multidrug resistance, even early in their hospitalizations [2,4,14,15]. K. variicola was more likely to be identified from BSIs and invasive disease. Further work is needed to help clinicians in interpreting the clinical significance of K. variicola. Larger Klebsiella isolate repositories that could be analyzed for resistance patterns, clinical characteristics, and for misidentification of K. variicola would be helpful in this regard. More phylogenetic work expanding our understanding of the genus Klebsiella is vital to further characterizing the role of specific species and their relevant pathogenic characteristics.

Raw images for pulsed-field gel electrophoresis analysis of 10 K. variicola isolates collected from wounded military personnel.

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13 May 2021

PONE-D-21-10025

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients

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[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 through a cooperative agreement with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). This project has been funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, https://www.niaid.nih.gov/, under Inter-Agency Agreement Y1-AI-5072 to DRT, the Defense Health Program, U.S. DoD, under award HU0001190002 to DRT, the Department of the Navy under the Wounded, Ill, and Injured Program (HU0001-10-1-0014) to DRT, and the Military Infectious Diseases Research Program, https://midrp.amedd.army.mil/ (HU0001-15-2-0045) to KM.  The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.].

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Reviewer #2: Yes

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Reviewer #1: The comments are attached. The comments for the paper " Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive K. variicola in trauma patients". There are comments and observations made which the authors are requested to address before publication

Reviewer #2: Dear Authors

The paper describes the speciation of Klebsiella variicola spp among previously isolated from trauma patients and identified as Klebsiella pneumoniae along with the resistance patters and clinical outcomes. While epidemiological studies like this throws so much light on speciation and outcomes of infections in patients with trauma especially in army veterans returning from Afghanistan, the authors may consider the following to improve the manuscript.

1. The authors have included isolates that were colonizers and those from infections. However, the time line of the colonization and if they had any clinical significance to go on and cause infection does not seem clear. How long ago from the posting in Afghanistan did they colonize with the isolates? Were these isolates fond before the posting or after the posting? Did the colonizers have any relevance to the isolates in the wound swabs of the patients?

2. Why did the authors stick to identifying only 3 species when Klebsiella pneumoniae , Klebsiella quasipneumoniae subsp. quasipneumoniae , Klebsiella quasipneumoniae subsp. similipneumoniae , Klebsiella variicola subsp. variicola , Klebsiella variicola subsp. tropica , Klebsiella quasivariicola , Klebsiella africana have been described?

3. Would the authors opine that these infections and colonizations were health care associated/ health care acquired infections?

4. Why was PCR not used to differentiated the hipervirulent isolates? String test seems to be a very crude method. Further, data on hypermucoviscosity vs hypervirulence may be a great add on.

5. What were the number of ESBL's detected in this population of isolates studied? What was the method employed to classify them as ESBL's? Which CLSI guideline was used ( Version)

6. Isolates from clinical samples need to be detailed if they were associated with healthcare associated infection. Were wound swabs the main source. If so were the wound sites in the groin region? If so were the same isolates found as colonizers and then went on to cause infections? The percentages of isolates and their distribution may also be shown in a figure. However, the percentage of K. variicola seems too small to draw any conclusions especially when isolated from the blood culture

7. Since the study period is between 2009-2014, in 5 years were there changes in the isolation rates? Maybe a timeline of the distribution of isolates in a figure would be useful?

**********

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Submitted filename: D-21-10025 MAY 2021 RESISTANCE PATTERNS OF K. PNEUMONIAE AND VARIICOLA IN TRAUMA PATIENTS.docx

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14 Jun 2021

Additional Editor Comments:

Two reviewers have commented on the manuscript and raised important questions about the methodology used for antimicrobial sensitivity testing, to characterise hypervirulent strains, differentiation of colonisers from those causing infection, species of Klebsiella considered and other aspects of the manuscript. Please address all points raised by the reviewers and revise the manuscript.

Author Response: Thank you for the opportunity to revise. We have reviewed and comments from the reviewers and made changes to the manuscript accordingly.

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[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 through a cooperative agreement with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF). This project has been funded by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, https://www.niaid.nih.gov/, under Inter-Agency Agreement Y1-AI-5072 to DRT, the Defense Health Program, U.S. DoD, under award HU0001190002 to DRT, the Department of the Navy under the Wounded, Ill, and Injured Program (HU0001-10-1-0014) to DRT, and the Military Infectious Diseases Research Program, https://midrp.amedd.army.mil/ (HU0001-15-2-0045) to KM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.].

We note that one or more of the authors are employed by a commercial company: Advancement of Military Medicine, Inc.

1. Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Author Response: The company you are referencing is the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., which is a non-profit authorized by Congress to support research at the Uniformed Services University. The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc., is directly referenced in the submitted Funding Statement that was supplied during the initial submission “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 through a cooperative agreement with The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF).” The text has been revised to reference authors who received salaries from HJF. “Support in the form of salaries was provided by HJF for authors KM, SJK, MLC, and DL; HJF did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

We reviewed the author contribution section and no changes are needed.

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Author Response: The Competing Interests Statement has been revised to be the following: “KM, SJK, MLC, and DL are employees of the Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. (HJF), a not-for-profit Foundation authorized by Congress to support research at the Uniformed Services University of the Health Sciences (USU) and throughout military medicine. This does not alter our adherence to PLOS ONE policies on sharing data and materials. Please see Data Availability Statement.”

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Reviewer #1: Partly

Reviewer #2: Yes

________________________________________

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Reviewer #1: Yes

Reviewer #2: N/A

________________________________________

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Reviewer #2: Yes

________________________________________

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Reviewer #1: The comments are attached. The comments for the paper " Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive K. variicola in trauma patients". There are comments and observations made which the authors are requested to address before publication

An unique article detailing the incidence of infections and colonization by Klebsiella species, particularly K. variicola which may be misidentified as K. pneumoniae. The authors have selected trauma patients among the army veterans who participated in the battle in Afghanistan and were supposedly hospitalized and underwent procedures for various indications

The article is well presented and thoughts have been collated to bring out the said manuscript. The following observations are in order:

1. Colonisation was defined through the study to reflect those isolates obtained from groin surveillance swabs. Any particular reason for doing so, as Klebsiella spp are known to colonise a number of body sites including the respiratory and urinary tract without causing clinical infections. Can the authors substantiate this criteria for selection site for surveillance of colonization

Author Response: Thank you for the comment. As you rightly point out, Klebsiella spp. are known to colonize many organ systems. As part of infection control measures to identify patients with skin colonization of a wide variety of organisms and to limit transmission of multidrug-resistant organisms, surveillance cultures were collected at admission to the military hospitals (groin/axillary swabs at Landstuhl and groin/axillary/nares swabs at U.S.-based hospitals). The collection of the admission surveillance swabs was per policies at each of the sites and not based on instructions from TIDOS investigators. The sentence defining colonization in the Methods text (lines 93-95) was revised to clarify this point and now reads “Colonization was defined as recovery of isolate from groin swabs obtained as part of targeted infection control surveillance at hospital admission for their deployment-related injury.”

2. Klebsiella isolate analysis: The authors have alluded to the fact that Vitek and BD systems are unable to differentiate between K. pneumoniae, variicola and quasipneumoniae. Why have the authors chosen just three species for ability or inability of the systems for species differentiation. What may be the reason for this specific focus on the said three species and not the others

Author Response: Thank you for the comment. At the time of the study development, we had elected to utilize the PCR technique described by Garza-Ramos and colleagues (BMC Microbiol. 2015; 15:64; BMC Microbiol. 2016; 16:43) that would be able to distinguish these three species, primarily because of the ability to identify K. variicola. This decision hinged on our specific clinical question raised by the Sweden bacteraemia data (Maatallah et al. 2014. PLoS One. 9(11): e113539) and further discussed/elucidated by Garza-Ramos and colleagues. Since the laboratory bench work on our project was completed, there has continued to be literature published in the area of the phylogenetic and epidemiologic nature of the Klebsiella genus, leading ultimately to the elucidation of five different species (including here K. quasivariicola). Given this evolution in the landscape of the genus, we added a new reference as #9 (Barrios-Camacho H, et al. Sci Rep. 2019; 9(1): 10610) to the Introduction and clarifying language to the discussion of this manuscript on page 17 (lines 336-338) to give the reader a greater sense of the scope of this work and recent developments that have taken place in the scientific community.

Does literature suggest that there aren’t other species of Klebsiella that could prove pathogenic other than the ones tested for here in this article?

Author Response: Thank you for the comment. There has been literature published (or is in pre-print) since we completed our study that indicates the occurrence of other species of Klebsiella that have specific pathogenicity associated with them, in particular the association with K. variicola as a uropathogen and hypervirulent strains of K. quasipneumoniae. The recently described K. africanensis and K. quasivariicola would also deserve attention for future work attempting to further fill in the details of the pathogenicity of the entire complex. As above, we have alluded to this evolving information with edits in lines 336-338.

3. The same (? bias) is reflected in this subsequent section on PFGE of the isolates where the PCR method and the primers used were dictated by the choice of the species rather than a broad range of species. It may have been useful to carry out the PCR to be able to identify other unusual species of Klebsiella too. Can this be substantiated by the authors?

Author Response: We agree that broad range PCR would have given additional data. Based off what we knew from the published literature at the time of the initiation of the analysis, we felt a more targeted approach, particularly of a clinical cohort of patients would be better suited to try to answer the specific question we had on K. variicola.

4. Virulence factors for Hypervirulence in Klebsiella species is best done with a genetic analysis of the isolates where there is an attempt made to amplify the presence of genes such as mag A gene ( for instance) String test is a crude method and is not acceptable for drawing conclusion on the possible hypervirulence of Klebsiella species .

Author Response: We completely agree that the string test is a crude test and clarified the text in the Methods (the choice of using this bench side test; page 6, lines 131-133), as well as in the Discussion on page 16 (lines 311-312) to emphasize the careful distinctions made between virulence and hyperviscosity made by Catalan-Najera and colleagues in Virulence (2017; 8[7]:1111-1123) regarding this important point.

5. Of the 51 infecting Klebsiella isolates, 16 were from the respiratory tract. Can the authors furnish information/ data on whether these respiratory infections were Lower respiratory tract infections, VAP, HAP etc. This is important as Klebsiella is known to colonise the respiratory tract of hospital and facility in- patients. The same holds good for the 5 isolates from the urinary tract.

Author Response: Thank you for the question. These were lower respiratory tract infections – the way the initial data collection was done, the isolates were definitional not colonizers. Timing of infections was also part of the data collection, but not explicitly identified as VAP. Urinary isolates were considered associated with infection as defined in the methods (lines 95-96). We have since clarified the text in the Methods (lines 93-96) to reflect that only isolates collected from groin swabs were considered colonizers, and all remaining clinical isolates included were considered infecting.

6. Isolate analysis: The fourth line in the 2nd paragraph talks about substantial resistance to ESBL inhibitors. Can the authors qualify this please, as one only alludes to Beta lactam lactamase inhibitors in scientific literature. The only inhibitor visible in Table -4 was Piperacillin tazobactam and this is not classified scientifically as an ESBL inhibitor

Author Response: Thank you for the comment and we agree. We have updated the language on page 12 (line 225) to specify piperacillin-tazobactam.

7. There was a mention in the Materials and methods section of classifying isolates as MDR based on resistance / ESBL/ Carbapenamase etc. However the same is not reflected in the Results section as to how many of the isolates showed ESBL, derepressed Amp C or a Carbapenamase enzyme. This may be an useful information in a manuscript dealing with resistance patterns and related clinical outcomes

Author Response: Thank you for your comment and we agree. Ultimately, we had very few CRE organisms, and we did not have the bench work to support genetic evidence for specific cases of suspected Amp C derepression, so we felt that for comparing groups, our analysis would be strongest by grouping the mechanisms into multidrug resistance. We have included text on page 17 (lines 334-335), fully acknowledging this limitation.

8. Klebsiella variicola: in the Results section: The authors have stated that 80% of the K. variicola isolates were from blood cultures. 4 of the 5 isolates does not qualify for a percentage. The same concept is again represented in the discussion section.

Author Response: Thank you for your comment. We have revised the sentence in the Results to remove the 80%, so it is a statement of numbers only. The statement in the Discussion was revised to be the following: “The second notable point is that four of the five infecting K. variicola isolates were identified from blood.”

We also adjusted a sentence in the abstract that referenced the data to now be “Compared to K. pneumoniae, infecting K. variicola isolates were more likely to be from blood (4/5 versus 24/134, p=0.04), and less likely to be multidrug-resistant (0/5 versus 99/134, p<0.01).”

9. Discussion section Page 14: The last two lines taken about the “overall proportion of misidentified blood stream isolates 14% of 28 in our study”. this statement needs clarification as this does impact the discussion and the concluding paragraph.

Author Response: Thank you for your question. There were 28 BSI isolates initially identified as K. pneumoniae, but 4 were later classified as K. variicola. The text has been revised for clarity to read “Nevertheless, the overall proportion of misidentified K. variicola bloodstream isolates (4 isolates misidentified; 14% of 28) in our study is comparable to these prior published reports.” The goal of this language was to compare our reported misidentification in bloodstream isolates to the other published reports.

Reviewer #2: Dear Authors

The paper describes the speciation of Klebsiella variicola spp among previously isolated from trauma patients and identified as Klebsiella pneumoniae along with the resistance patters and clinical outcomes. While epidemiological studies like this throws so much light on speciation and outcomes of infections in patients with trauma especially in army veterans returning from Afghanistan, the authors may consider the following to improve the manuscript.

Author Response: Thank you for your comments

1. The authors have included isolates that were colonizers and those from infections. However, the time line of the colonization and if they had any clinical significance to go on and cause infection does not seem clear. How long ago from the posting in Afghanistan did they colonize with the isolates? Were these isolates fond before the posting or after the posting? Did the colonizers have any relevance to the isolates in the wound swabs of the patients?

Author Response: Thank you for your comments. The colonizing isolates were recovered as part of infection control procedures (groin surveillance swabs) at hospital admission for their deployment-related injury following medical evacuation from Afghanistan. The sentence defining colonization in the Methods text (lines 93-95) was revised to clarify this point and now reads “Colonization was defined as recovery of isolates from groin swabs obtained as part of targeted infection control surveillance at hospital admission for their deployment-related injury.” In addition, the sentence (lines 110-111) that described the linking of infecting isolates to colonizing isolates was revised to “All colonizing isolates linked with infecting isolates (defined as isolation from groin admission swab prior to infection) were included.”

2. Why did the authors stick to identifying only 3 species when Klebsiella pneumoniae, Klebsiella quasipneumoniae subsp. quasipneumoniae, Klebsiella quasipneumoniae subsp. similipneumoniae, Klebsiella variicola subsp. variicola, Klebsiella variicola subsp. tropica, Klebsiella quasivariicola, Klebsiella africana have been described?

Author Response: Thank you for the comment. Essentially, we felt that one of the questions we had pre-specified before doing this work was to focus on the role of K. variicola that had been published in the literature and that our N and subsequent design of the bench work didn’t allow us to broadly PCR for the rest of the species within the genus –as you rightly point out. Please also see the response to comment #2 from Reviewer #1.

3. Would the authors opine that these infections and colonizations were health care associated/ health care acquired infections?

Author Response: Thank you for the question—we have added a sentence that clarifies the nosocomial nature of these isolates within the Discussion (lines 274-276), prior to a sentence that discusses the essential role of infection prevention and control as well as antimicrobial stewardship in decreasing their impact.

4. Why was PCR not used to differentiated the hipervirulent isolates? String test seems to be a very crude method. Further, data on hypermucoviscosity vs hypervirulence may be a great add on.

Author Response: Thank you for your comment and we completely agree. We felt that this would be a question that many readers might have (the string test), but ultimately after this crude screening test, felt that beyond reporting the data, there were not any additional conclusions to be drawn. A statement was added to the Discussion on page 16 (lines 311-312), which states “It is important to note here that the lack of the hypermucoviscous phenotype does not necessarily exclude a particular isolate from the hypervirulent phenotype” and as above, added the reference by Catalan-Najera and colleagues in Virulence (2017; 8[7]:1111-1123) regarding this important point.

5. What were the number of ESBL's detected in this population of isolates studied? What was the method employed to classify them as ESBL's? Which CLSI guideline was used ( Version)

Author Response: ESBL’s were not explicitly re-identified outside of the automated breakpoint data that accompanies the BD Phoenix. The CLSI guideline used was M100, 28th edition published in 2018 and that information was added to line 119 on page 6 in the Methods section.

6. Isolates from clinical samples need to be detailed if they were associated with healthcare associated infection. Were wound swabs the main source. If so were the wound sites in the groin region? If so were the same isolates found as colonizers and then went on to cause infections? The percentages of isolates and their distribution may also be shown in a figure. However, the percentage of K. variicola seems too small to draw any conclusions especially when isolated from the blood culture

Author Response: Thank you for your comments. The text in the Results Isolate Analysis section was revised to clarify the number of K. pneumoniae infecting isolate sources. The revised sentence reads “Sources of the 134 K. pneumoniae infecting isolates were wound (N=57, 43%), respiratory (N=29, 22%), blood (N=24, 18%), intraabdominal (N=4, 3%), and other (N=20, 15%).” Text in the Klebsiella variicola section was also revised to clarify the sources of the infecting isolates and now reads “Five isolates were colonizers from groin swabs, while the five isolates associated with infections were from blood (N=4), and intraabdominal specimens (N=1).”

Yes, wound swabs were the main source for K. pneumoniae, but it was blood for K. variicola. Wound sites could derive from the abdomen, gluteal, hand, lower leg, pelvis, thigh, and upper arm. Additionally, we further clarified within the methods (as discussed in response to reviewer 1) the definitions of colonizing vs infecting isolates.

7. Since the study period is between 2009-2014, in 5 years were there changes in the isolation rates? Maybe a timeline of the distribution of isolates in a figure would be useful?

Author Response: Thank you for the comment. We did have similar questions about isolation rates, particularly when it comes out in outbreak analyses; however, outside of our outbreak analysis where there were important features of timing, we thought that battlefield kinetics and timing of injuries would be hard to control for. Text related to the number of isolates collected over the study years was added to the Results on page 11, lines 214-219. The new text states that ‘The years of K. pneumoniae collection were 28 (13% of 221) isolates in 2009, 95 (43%) in 2010, 30 (14%) in 2011, 51 (23%) in 2012, 13 (6%) in 2013, and 4 (2%) in 2014. There were no K. variicola isolates collected in 2009, while three were collected in 2010, four were collected in 2011, and one isolate was collected per year from 2012-2014. Four of the K. quasipneumoniae isolates were collected in 2011, one isolate in 2013, and one isolate in 2014.’

We have also added a sentence to the Discussion (lines 324-326) to discuss how the number of isolates tapering off likely reflects the decreasing numbers of evacuated casualties towards the end of the study period, coinciding with the end of combat operations in Afghanistan.

Submitted filename: Response to Reviewers.docx

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21 Jul 2021

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients

PONE-D-21-10025R1

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Comments to the Author

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: All previous queries raised have been adequately addressed . All specific questions have been answered by the authors

Reviewer #2: Dear authors.

All comments have been adequately addressed and adequate references have been added to substantiate the explanation.

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Reviewer #1: No

Reviewer #2: Yes: Anusha Rohit

23 Jul 2021

PONE-D-21-10025R1

Resistance patterns and clinical outcomes of Klebsiella pneumoniae and invasive Klebsiella variicola in trauma patients

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