Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer.

OBJECTIVES: Multidrug-resistant organisms (MDRO) are considered an emerging threat worldwide. Data covering the clinical impact of MDRO colonization in patients with solid malignancies, however, is widely missing. We sought to determine the impact of MDRO colonization in patients who have been diagnosed with Non-small cell lung cancer (NSCLC) who are at known high-risk for invasive infections.
MATERIALS AND METHODS: Patients who were screened for MDRO colonization within a 90-day period after NSCLC diagnosis of all stages were included in this single-center retrospective study.
RESULTS: Two hundred and ninety-five patients were included of whom 24 patients (8.1%) were screened positive for MDRO colonization (MDROpos) at first diagnosis. Enterobacterales were by far the most frequent MDRO detected with a proportion of 79.2% (19/24). MDRO colonization was present across all disease stages and more present in patients with concomitant diabetes mellitus. Median overall survival was significantly inferior in the MDROpos study group with a median OS of 7.8 months (95% CI, 0.0-19.9 months) compared to a median OS of 23.9 months (95% CI, 17.6-30.1 months) in the MDROneg group in univariate (p = 0.036) and multivariate analysis (P = 0.02). Exploratory analyses suggest a higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p = 0.002) with an increased rate of fatal infections in MDROpos patients (p = 0.0002).
CONCLUSIONS: MDRO colonization is an independent risk factor for inferior OS in patients diagnosed with NSCLC due to a higher rate of fatal infections. Empirical antibiotic treatment approaches should cover formerly detected MDR commensals in cases of (suspected) invasive infections.

Introduction

Multidrug-resistant organisms (MDRO) such as vancomycin-resistant Enterococci (VRE), third-generation Cephalosporin-resistant Enterobacterales, piperacillin/tazobactam-resistant Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus (MRSA) are considered an emerging threat worldwide as there are fewer and sometimes even no antimicrobial agents left to treat infections caused by these pathogens [1, 2]. The impact of MDRO in patients with hematologic malignancy has been investigated extensively [3-6]. Hematologic patients colonized with MDRO are at a profound risk of invasive MDRO infections [7-9]. Infections with MDRO provoke prolonged hospital stays, increased hospital costs and negatively impact survival [3, 10–14].

However, only few clinical studies have addressed the impact of MDRO infections (compared to non-MDRO infections) in patients with solid malignancies. As most of these analyses suffer from several limitations such as focusing solely on critically ill patients treated on intensive care units [11, 15, 16], providing only short-term follow-ups [17] or including various oncological entities at different disease stages [18], valid conclusions on the overall survival impact of colonization and infection with MDRO in patients with solid malignancies cannot be drawn.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide [19]. Most patients are diagnosed in advanced disease stages and palliative treatment choices consist of targeted therapy, immunotherapy and cytotoxic agents such as platinum–based chemotherapy. Patients at all stages are at high risk of life-threatening infections due to invasive therapeutic and diagnostic procedures, immunocompromising therapy and related comorbidities (e.g. chronic obstructive pulmonary disease) [20]. Large prospective clinical trials report bacterial infection rates in approximately 10% of patients with limited or advanced disease stages [21-27] and up to 70% in retrospective analyses [28, 29]

The presence of MDRO colonization in patients with NSCLC and its impact on survival has not been investigated so far. We therefore sought to determine the frequency, clinical characteristics and clinical impact with a focus on survival outcomes of MDRO colonization in patients with NSCLC in a retrospective single center analysis.

Material and methods

Defining the study population

Patients diagnosed with NSCLC, stages I-IV according to the Union International Contre le Cancer (UICC) 7th edition between 2012 and 2016 and screened for MDRO (definition see below under “screening procedures and definitions”) within a time period of 90 days calculated from pathological confirmed first diagnosis of NSCLC were included to this analysis. Exclusion criteria were history of or concomitant underlying second malignancy—aside from localized non-melanoma skin cancer (e.g. basalioma) that had been curatively treated -, insufficient case documentation and missing MDRO screening. Patient data used in this study were provided by the University Cancer Center Frankfurt (UCT). Written informed consent was obtained from all patients and the study was approved by the institutional Review Boards of the UCT and the Ethical Committee at the University Hospital Frankfurt (project-number: STO-01-2016, Amendment 1, 06.06.2018).

Screening procedure and definitions

According to German infection law (Infektionsschutzgesetz, IfSG) [30] execution of an infection control protocol in order to prevent the transmission of infective agents, such as MDRO is mandatorily required. At the University hospital Frankfurt, this legal requirement by IfSG as well as the recommendations of the Commission for Hospital Hygiene and Infection Prevention (KRINKO) at the Robert Koch Institute, Berlin, Germany (e.g. recommendations for prevention and control of MRSA in medical and nursing facilities; [31]) are entirely fulfilled. Therefore, patients reporting defined risk factors, e.g. arriving from high-prevalence countries, e.g. including but not limited to countries from the middle east, south-east Asia and India for MDRO, being refugee as well as patients admitted to any intensive/intermediate care unit as well as all patients admitted to the thoracic surgery ward and patients admitted to the clinical oncology ward need to be screened for MDRO at the day of admittance by nasal, rectal and pharyngeal swabs [32, 33].

MDRO were defined as Enterococcus faecium or Enterococcus faecalis with vancomycin resistance (VRE) and Methicillin-resistant Staphylococcus aureus (MRSA). Multidrug-resistant gram-negative bacteria were defined as Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Proteus mirabilis with extended spectrum beta–lactamase (ESBL)–like phenotype as well as Enterobacterales, Acinetobacter baumannii and Pseudomonas aeruginosa resistant against piperacillin, any 3rd/4th generation Cephalosporin, and fluoroquinolones ± carbapenems [31].

Patients were defined as “colonized” if an MDRO was detected (MDROpos) in at least one nasal, rectal or pharyngeal swab. Screened patients without evidence of MDRO colonization were defined as MDROneg. In case of multiple MDRO screenings within the predefined time period at first diagnosis, the first screening result defined group assignment.

Detection and molecular resistance analysis in MDRO

Rectal swabs were collected using culture swabs with Amies collection and transport medium (Hain Lifescience, Nehren, Germany) and were afterwards streaked onto CHROMagarTM ESBL plates (Mast Diagnostica, Paris, France), chromID CARBA (bioMérieux, Nürtingen, Germany), chromID VRE (bioMérieux), chromID OXA-48 (bioMérieux), Brilliance MRSA-Agar (Oxoid, Wesel, Germany). Matrix-assisted-laser desorption ionization-time of flight analysis (MALDI–TOF) and VITEK2 (bioMérieux) were used to identify gram negative species, when growth was detected. Antibiotic susceptibility testing was carried out according to the Clinical and Laboratory Standards Institute (CLSI) guidelines by VITEK 2 and antibiotic gradient tests (bioMérieux) or agar diffusion (Oxoid). Carbapenemase encoding genes were detected via polymerase chain reaction analysis and subsequent sequencing from carbapenem-resistant Enterobacterales including the bla genes for carbapenemases OXA–48, OXA–48 like and KPC, NDM, VIM, IMP as well as OXA–23, OXA–24, OXA–51, and OXA– 58 for A. baumannii [34]. For the detection of MRSA, nasal and pharyngeal swabs were inoculated on Brilliance MRSA Agar (Oxoid, Wesel, Germany). Identification of MRSA species was done by MALDI–TOF and antibiotic susceptibility testing using VITEK 2. The clonal identity of MRSA isolates was analyzed by staphylococcal protein A (spa) typing using the Ridom StaphType software (Ridom GmbH, Würzburg, Germany), as previously reported [32, 34]. All laboratory testing was performed under strict quality-controlled criteria (laboratory accreditation according to ISO 15189:2007 standards; certificate number D–ML–13102–01–00, valid through January 25th, 2021).

Study endpoints

Predefined primary study endpoints were event-free-survival (EFS) and overall survival (OS) compared between MDROpos and MDROneg groups, taking into account known confounding variables such as gender, age, disease stage, Eastern Cooperative Oncology Group (ECOG) Performance Status, NSCLC histology, smoking status and concomitant diseases in multivariate analysis. Event-free-survival was defined as the time period until re-occurrence of histologically confirmed lung cancer after curative treatment or the time period until next treatment line or death from any cause, whichever came first. Patients who were still alive at data cut-off were censored with regard to OS at the date of last contact. Patients who did not die or did not show any of the above-mentioned events at the time of the data cut-off were censored with regard to EFS analysis at the date of last contact.

Secondary endpoints were the distribution of causes of death stratified by MDRO colonization status and number and length of hospital stays stratified by cause of inpatient treatments. The specific causes of death were extracted from the letter of notification or death certificate. Exploratory endpoints were the rate of subsequently detected invasive MDRO infections and evaluation of antibiotic approaches in MDROpos patients with infectious complications. Finally, we planned to compare the eligible study cohort to patients who were primarily excluded from the analysis due to missing MDRO screening (off-target population).

Statistical analysis

The number of all included patients and recorded variables were reported descriptively. Survival analyses were performed using the Kaplan-Meier method for estimation of the percentage of surviving patients and the log-rank test for comparing patient groups. Cox proportional hazard regression analysis was used for multivariate analyses. Proportional hazards assumption and residuals were checked formally and graphically. Schoenfeld residuals for all covariates were verified to be independent of time. Competing risks of death and their cumulative incidences were analyzed using R’s cmprsk package implementing the proportional subdistribution hazards’ regression model described in Fine and Gray (1999) [35] with failure types as indicated and MDRO colonization as a binary covariate. Comparative analyses for differences in proportions and other numerical variables between study groups were performed using Chi2 test and Mann-Whitney U test. A p-value below 0.05 was considered statistically significant. R version 3.5.1 and GraphPad Prism version 6.01 were used for statistical analysis and reporting of the data collected for this study.

Results

Study population and off-target analysis

We identified 639 patients diagnosed with NSCLC between 2012 and 2016 in the institutional cancer registry of the University Hospital, Frankfurt am Main, Germany of whom 295 were available for further analysis. A CONSORS flow chart showing the process of inclusion of eligible patients into the analysis is available in S1 Fig in S1 File. Twenty-four patients (8.1%) were colonized with MDRO (MDROpos). Two hundred seventy-one patients (91.9%) were defined as MDROneg within the screening period. Median time to first MDRO screening calculated from first diagnosis was 20 days (range, 0–84 days). Comparative descriptive statistics of the study groups are illustrated in Table 1. Median age was 66 years (range, 29–90 years). Approximately 80% had an ECOG performance status of 0 or 1 and one third of all patients presented with metastatic disease stage (UICC IV). The majority of patients were former or active smokers. Aside of concomitantly underlying diabetes mellitus that was more frequently present in MDROpos patients, we did not find significant differences in patient or disease characteristics between MDROpos and MDROneg patients in univariate and multivariate analysis (S2 Table in S1 File).

Table 1

Patient and disease characteristics.

		All patients (n = 295)	No MDRO (n = 271)	MDRO colonization (n = 24)	P value*
Gender	female	110 (37%)	106 (39%)	4 (17%)	.05
	male	185 (63%)	165 (61%)	20 (83%)	.05
Age at diagnosis, median (range), years		67 (29–90)	67 (29–90)	70 (53–90)	.17
Smoking history		209 (71%)	192 (71%)	17 (71%)	1
ECOG performance score ≤ 2 (%)		281 (95%)	259 (96%)	22 (92%)	.72
UICC	IA	50 (17%)	45 (17%)	5 (21%)	.81
	IB	19 (6%)	18 (7%)	1 (4%)	.97
	IIA	22 (7%)	21 (8%)	1 (4%)	.81
	IIB	25 (9%)	24 (9%)	1 (4%)	.68
	IIIA	61 (21%)	54 (20%)	7 (29%)	.42
	IIIB	19 (6%)	19 (7%)	0 (0%)	.36
	IV	99 (34%)	90 (33%)	9 (38%)	.84
Presence of brain metastases		48 (16%)	47 (17%)	1 (4%)	.17
Histology	Adeno NSCLC	160 (54%)	150 (55%)	10 (42%)	.28
	SCNSCLC	127 (43%)	113 (42%)	14 (58%)	.17
	other	8 (3%)	8 (3%)	0 (0%)	.84
Mutations (pos. / neg.)	ALK	3 (1%) / 32 (11%)	2 (1%) / 29 (11%)	1 (4%) / 3 (13%)
	BRAF	2 (1%) / 5 (2%)	1 (0%) / 4 (1%)	1 (4%) / 1 (4%)
	EGFR	15 (5%) / 33 (11%)	15 (6%) / 30 (11%)	0 (0%) /3 (13%)	.45
	KRAS	15 (5%) / 14 (5%)	13 (5%) / 13 (5%)	2 (8%)/ 1 (4%)
	ROS1	4 (1%) / 12 (4%)	3 (1%) / 11 (4%)	1(4%) / 1 (4%)
Comorbidities	Diabetes	56 (19%)	44 (16%)	12 (50%)	.0002
	HIV	9 (3%)	9 (3%)	0 (0%)	.77
	Heart disease	60 (20%)	53 (20%)	7 (29%)	.39
	Kidney disease	52 (18%)	45 (17%)	7 (29%)	.21
	Liver disease	9 (31%)	8 (3%)	1 (4%)	1
1st line treatment approach	Surgery only	69 (23%)	63 (23%)	6 (25%)	.85
	Surgery + adjuvant / neoadjuvant platinum based CTX	101 (34%)	93 (34%)	8 (33%)	.85
	RCTX	11 (4%)	11 (4%)	0 (0%)	.31
	Target Therapy	4 (1%)	3 (1%)	1 (4%)	.21
	Platinum-based CTX	95 (32%)	87 (32%)	8 (33%)	.68
	Other	5 (2%)	5 (2%)	0 (0%)	.50
	BSC	5 (2%)	5 (2%)	0 (0%)	.50
	Unknown	5 (2%)	4 (1%)	1 (0%)	.35

Count data is shown unless indicated otherwise. *Differences between colonized and non-colonized patients were tested. Mann-Whitney U test was used to calculate P value for age. Except for EGFR, gene mutations were not tested due to missing data. CT, chemotherapy; TKI, tyrosine kinase inhibitor; NSCLC, non- small cell lung cancer; SCNSCLC, squamous cell NSCLC.

First-line treatment approaches did not differ significantly between study groups. Notably, only a minority of patients diagnosed with driver mutations received a first-line targeted therapy. This is partially owed to the fact that ALK, ROS1 and BRAF inhibitors were first approved for first line treatment in Germany in late 2016 and 2018, respectively. Five patients in the MDROneg group and no patient in the MDROpos group received best supportive care only.

We then compared the eligible study cohort with patients identified in the registry without MDRO screening within the predefined time frame. The off-target population (107/402; 26.6%) was significantly younger (p = 0.001), had a higher proportion of patients with ECOG 3 or worse performance status in addition to a higher proportion of patients with advanced or metastatic disease (p = 0.0001) (S3 Table in S1 File). Besides diabetes, which was more prevalent in the study cohort (p = 0.004), other comorbidities were well balanced. The OS of the off-target cohort was significantly inferior compared to the study cohort, yet no survival differences in patients with advanced or metastatic disease (IIIB, IV; UICC 7th) between the overall off-target and the study population were noticed (not shown).

MDRO

A total of 24 patients (8.1%) were screened positive for MDRO colonization. Detailed information on resistance phenotype of all MDRO is shown in S4 Table in S1 File. Enterobacterales were by far the most frequent MDRO detected with a proportion of 79.2% (19/24), all of which had phenotypical resistance to 3rd/4th generation Cephalosporins (Ceftriaxone, Cefotaxime, Ceftazidime, Cefepime). Additionally, most species were resistant to piperacillin and more than half were resistant to folate pathway inhibitors (Trimethoprim/Sulfamethoxazole). Resistance against aminoglycosides (Amikacin, Gentamicin), tigecycline and fosfomycin were infrequent. All MDR Enterobacterales detected were susceptible to carbapenems (Imipenem, Meropenem, Ertapemem). Enterococcus faecium with resistance to ampicillin, carbapenem and fluoroquinolones (Levofloxacine, Ciprofloxacine, Moxifloxacine) and incomplete resistance to glycopeptides (Vancomycine, Teicoplanin)(3x vanB phenotype, 1x vanA phenotype) were detected in 16.7% (4/24) of all MDROpos cases. Additional resistance to aminoglycosides (high-level) and tetracyclines was detected in one case each. One MRSA (4.2%, 1/24) with phenotypical resistance against fluoroquinolones, lincosamides (Clindamycin) and macrolides (Erythromycin) was identified. The most common location for MDRO colonization was rectal (95.8%) in all but the MRSA case, which was detected in a nose swab.

The incidence of subsequent colonization with multiple MDRO in MDROpos patients within the screening period was 25%, 3 patients acquired additional ESBL-producing species and 3 patients acquired additional VRE. Altogether, 16 patients in the MDROneg group were subsequently screened positive for MDRO after a median time calculated from first diagnosis of 495 days (range, 109–1231 days). Because subsequent screening procedures in patients with NSCLC were only irregularly performed, especially in patients who were mainly treated on an outpatient basis, further analyses on these patients (with subsequently acquired MDRO colonization) were not carried out due to probable selection bias of this subpopulation.

Primary outcome analysis: Survival

Kaplan-Meier estimates for EFS and OS of the overall population and stratified by MDRO colonization are shown in Fig 1A–1D. Median EFS did not differ between MDROpos (7.1 months; 95% CI, 0.0–16.7 months) and MDROneg (10.3 months; 95% CI, 7.9–12.9 months) study groups with a hazard ratio (HR) of 1.25 (95% CI, 0.74–2.21; p = 0.25) (Fig 1D), that was further confirmed by multivariate analysis (S5 Table in S1 File). There were 92 censored events (31.2%) in the EFS analysis. Median OS was significantly inferior in the MDROpos study group with a median OS of 7.8 months (95% CI, 0.0–19.9 months) compared to a median OS of 23.9 months (95% CI, 17.6–30.1 months) in the MDROneg group resulting in a HR of 1.9 (95% CI, 1.02–3.7); p = 0.036)(Fig 1B). There were 120 censored events (41.0%) in the OS analysis.

Fig 1

Kaplan-Meier estimates of overall survival (OS) and progression-free survival (PFS).

(A) OS of all patients. (B) OS of patients stratified by multidrug-resistant organism (MDRO) colonization. (C) PFS of all patients. (D) PFS of patients stratified by colonization with MDRO. Log-rank test was used to calculate p values in (C+D).

When stratified for disease stage (Fig 2A), median OS in the MDROpos study group showed a significantly inferior median survival time in patients with advanced (IIIB) or metastatic disease (IV)(4.4 months vs 10.5 months; HR, 2.9; 95%CI, 1.9–19.6; p = 0.0004) (Fig 2C), whereas we found no significant difference in survival between MDROpos and MDROneg study groups with early disease stages (IA-IIIA; HR 1.4; 95%CI, 0.6–3.5; p = 0.39)(Fig 2B). Stratification by MDRO species did not yield significant differences in OS among MDROpos patients colonized with VRE, MRSA or ESBL (p = 0.12) (Fig 2D). The negative impact on survival outcomes was further confirmed in multivariate analysis adjusted for gender, age, disease stage, ECOG performance status, NSCLC histology and presence of concomitant underlying diabetes (Fig 3). In addition to MDRO colonization, performance status and disease stage were identified as independent prognostic variables.

Fig 2

Competing risks analysis for death.

(A) Cumulative incidence functions for relapse mortality, non-relapse mortality, or mortality not otherwise specified (unknown) of all patients. (B) Cumulative incidence functions of patients stratified by multidrug-resistant organism (MDRO) colonization. Competing risks regression model [35] was used to calculate p values for differences in non-relapse mortality (p < 0.00001) and relapse mortality (p = 0.49) between patients colonized by MDRO and patients without MDRO.

Fig 3

Multivariate analysis of risk factors for death.

Secondary and exploratory outcome analysis

Cause of death

The distribution of causes of death stratified by MDRO colonization status is depicted in Table 2 and Fig 4. There was a significantly higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p = 0.002) and a significantly higher rate of infectious causes (p = 0.002) The most frequently observed infection-related cause of death was pneumonia with or without septicemia in 5 cases in the MDROpos group, 2 additional patients died of pleural empyema. The empirical antibiotic treatment approach in 5 of these patients consisted of agents that were primarily tested non-susceptible to the detected MDRO. Invasive infections from the formerly detected MDRO within the MDROpos group were determined in two cases (2/7, 28.6%) (VRE-positive blood culture of a patient with pneumonia-induced sepsis; evidence of ESBL in pleural empyema). In the remaining 5 patients the pathogenic organism could not be detected by serial blood cultures.

Table 2

Equal distribution of causes of death between the MRDOpos and MRDOneg subgroup.

		no MDRO (n = 271)	MDRO colonization (n = 24)	P value
NCR		22 (8%)	9 (38%)	.0002
infectious related		8 (36%)	7 (78%)	.0002
cardiovascular disease related		9 (41%)	2 (22%)	.24
major bleeding with fatal outcome		4 (18%	0 (0%)	1.0
Asphyxia		1 (5%)	0 (0%)	1.0
CR		107 (39%)	6 (25%)	.40
UKN		29 (10%)	1 (4%)	.49
LTFU		113 (41%)	8 (33%)

Causes of death were compared using fishers exact test. NCR, non-cancer related mortality; CR, cancer-related; UKN, unknown; LTFU, lost to follow up; MDRO Multi Drug Resistant Organism; assessed using Fisher’s Exact Test.

Fig 4

Cumulative incidence of death stratified by non-cancer related and cancer related mortality (A) in the whole study group. (B) stratified by MDRO colonization.

In the MDROneg study group, 8 patients (36%) succumbed to infectious complications, 4 of which had evidence of an invasive pathogen. One of these patients died of pneumonia-induced sepsis caused by a subsequently acquired (after the initial screening period) piperacillin- and carbapenem-resistant Pseudomonas aeruginosa, whose profile of resistance could not be considered at the time of initial antibiotic treatment.

Number and duration of hospital stays

Overall, there were no differences in number and duration of all-cause hospital admissions between MDROpos and MDROneg patients. Likewise, there were no differences in number and duration of hospital admissions for infectious complications between MDROpos and MDROneg study groups (S6 and S7 Figs in S1 File). Comparison of number and duration of inpatient treatments between study groups were however not adjusted for differences in median survival times between MDROpos and MDROneg patients.

Discussion

To our knowledge, this is the first study that aimed to determine the clinical impact of MDRO colonization in patients with NSCLC. We show that MDRO colonization is an independent risk factor for impaired overall survival, independent of confounding variables, such as performance status and disease stage.

Our study demonstrates considerable colonization rates (8.1%) with ESBL producing Enterobacterales and VRE species in patients with NSCLC across all subgroups in terms of age, stage, performance status and concomitant underlying (renal, heart, liver) diseases among other variables. We encountered a significantly higher co-occurrence of diabetes in patients screened positive for MDRO. Diabetes has previously been identified as a potential risk factor for MDRO colonization [36, 37] and subsequent bloodstream infections with intestinal bacteria due to disruption of the gut barrier [38, 39]. The overall prevalence of MDRO colonization at admission has been reported to be as high as 10% for ESBL producing Enterobacterales [40, 41], reaching a prevalence of 20% in specific patient subgroups [9], and 2% for VRE [42] in German tertiary care centers. The colonization rate in our study was slightly lower than previously reported. Colonization rates are known to be significantly influenced by the patient subgroups examined and other risk factors such as antibiotic and surgical pretreatment, proton pump inhibitor usage, travel habits, prior hospitalizations and country of origin [33, 40, 43–45]. These factors were not assessed in our study and might contribute to the lower prevalence of MDRO colonization seen in our cohort. Furthermore, as many patients are seen as outpatients (with less stringent screening), MDRO positive patients may be underreported.

Approximately 80% of non-cancer-related mortality in the MDROpos group was infection-related as extracted from the corresponding death certificates. We did not observe any differences in hospital admission rates and/or duration of inpatient treatment (for infectious or other causes) between MDROpos and MDROneg patients, suggesting that MDRO colonization by itself may not be a strong risk factor for the frequency of subsequent invasive bacterial infections in this patient cohort, but instead mediates a higher fatality rate due to more severe infectious complications. However, this data is hard to interpret. Firstly, the number of outpatient visits (e.g. for infectious complications) could not be analyzed due to insufficient documentation. Secondly, we do not have sufficient information on the final course of each individual patient to judge the contribution of infectious-related complications to the death of patients with progressive cancer. And thirdly, we cannot exclude a misclassification of the cause of death by the responsible physician.

Infections, particularly involving the lung tissue have been identified as a major cause of death in several retrospective studies [28, 29]. Patients with advanced disease stages were more prone to infectious complication and data suggests that they may adversely affect survival.

It has been shown that the increased fatality rate in MDROpos patients is at least partially attributable to inadequate empirical antibiotic treatment in case of invasive infections [17, 46]. Indeed, in 5 of the 7 fatal infections within the MDROpos cohort, the initial antibiotic regime did not take into account the prior proven MDRO colonization. Colonizing MDR bacteria were detected in 2 out of the 7 cases (29%) of pulmonary infections reported here. This is in agreement with previous reports on the overall low sensitivity regarding the detection of invasive pathogens by blood cultures [47]. Bacteremia is diagnosed in less than 10% by serial blood cultures of patients suffering from pneumonia despite clinical indications of bloodstream infections. Nevertheless, gut bacteria play a major role in NSCLC-associated lung tissue infections [48-50] and empirical antibiotic treatment should be selected considering intestinal MDRO bacteria.

There is emerging evidence that the gut microbiota affects systemic inflammation and immunity and there are multiple possible mechanisms linking microbiota to carcinogenesis, tumor outgrowth and metastases, altered metabolism, pro-inflammatory and impaired immune-response [51-53]. Almost all colonizing MDRO in our study have been identified by rectal screening. Susceptibility to and presence of intestinal MDRO has been linked to alterations in the gut microbiota with reduced bacterial diversity [54, 55], which in turn is associated with reduced tumor response to cytotoxic agents and immunotherapy in lung cancer [56-59]. This is also supported by reduced clinical benefit from immunotherapy after the usage of antibiotics in patients with NSCLC [56, 60]. In our study, however, first-line EFS was not different between MDROpos and MDROneg groups, indicating only minor–if any–influence of MDRO on response to conventional antineoplastic therapy. As immunotherapeutic agents were not approved for first-line treatment in NSCLC until 2017, we cannot draw conclusions regarding the impact of MDRO colonization on the treatment response to immunotherapeutic agents. Prospective studies are needed to further address the relevance of MDRO colonization and the impact of intestinal microbiota alterations on tumor response to immunotherapy and/or cytotoxic agents.

Finally, there is conflicting evidence, whether MDR bacteria have additional genomic content including factors known or supposed to be associated with increased virulence [61, 62]. Vancomyin-resistant E. faecium and ESBL-producing species have been shown to incorporate virulence factors in co-occurrence with genes for antibiotic resistance [63-68] and these factors might overall contribute to the higher mortality seen in MDROpos patients. However, since genetic analyses addressing the co-occurrence of virulence factors other than antibiotic resistance genes were not performed, we can only speculate on their influence on the overall mortality outcome in our study.

We fully acknowledge the limitations of a retrospective analysis conducted in a single tertiary treatment center. Significant differences between the study group and off-target population are indicative of selection bias due to MDRO screening. However, the proportion of patients excluded from the final analysis due to missing MDRO screening was only approximately 25% of the total screening population (patients with second malignancy excluded). Additionally, due the overall limited sample size our results need confirmation in larger series before drawing final conclusions regarding the impact of MDRO colonization in patients with oncological diseases. However, we believe that our findings corroborate available data collected in patients with (dominantly) hematologic malignancies that consistently show inferior survival outcomes in patients either with invasive MDRO infections or MDRO colonization [3, 5, 9, 12–14, 17, 69–71].

Conclusion

We conclude that MDRO colonization our population is an independent risk factor for inferior OS in patients diagnosed with NSCLC. Impairment Patients with advanced or metastatic disease seem to be at highest risk for impaired survival. Furthermore our data suggest, that a higher rate of non-cancer related mortality and infections in particular might contribute to the inferior survival in MDRO colonized patients. Given the high and rising rate of MDRO colonization in oncological patients, early and frequent screening is warranted in both outpatient and inpatient settings. Empirical antibiotic treatment approaches need to cover formerly detected MDR commensals in cases of (suspected) invasive infections.

More studies should elucidate the impact of MDRO colonization and intestinal bacterial diversity within the rapidly changing landscape of antineoplastic treatment options in patients with NSCLC.

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10 Aug 2020

PONE-D-20-09321

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

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Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: Yes

**********

5. 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: The manuscript titled 'Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer' tries to show the importance of multi-drug resistant organisms on survival and mortality of patients suffering from non-small cell lung cancer. The main limitation of this investigation is the use of a very small sample to draw an important/significant conclusion.

Major comments:

1. The study sample size is small, only 24 out of 295 (8%) patients were studied. The sample size must be significantly larger than this to draw any valid conclusion.

2. In Pg 2 Lines 42-46, the authors mention that 'There was a significantly higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p<0.001) with a trend towards an increased rate of fatal infections in MDROpos patients (p=0.05).

Also, Pg 11, Lines 231-232 states 'When stratified for disease stage (Fig. 2A), median OS in the MDROpos study group showed a significantly inferior median survival time in patients with advanced (IIIB) or metastatic disease (IV). Then again, in Pg 14, Line 302, the authors say ‘Approximately 80% of non-relapse mortality in the MDROpos group was infection-related.’

These are strong statements. Patients in advanced stages of cancer are more likely to suffer from mortality when compared to those in early stages of cancer simply because of their physiological conditions and not just because of bacterial colonization.

Bacterial infections in patients at early stage of cancer can actually augment progression to more critical stages/metastasis according to the existing literature. So, it is the severity of the infection at the early stage which determines or is one of the predisposing factors to progression to a more critical stage.

3. There are many existing literature that the authors can compare their work with such as:

i. Sarihan S, Ercan I, Saran A, Cetintas SK, Akalin H, Engin K. Evaluation of infections in non-small cell lung cancer patients treated with radiotherapy. Cancer Detect Prev. 2005;29(2):181-188. doi:10.1016/j.cdp.2004.11.001

ii. https://www.cancernetwork.com/view/infectious-complications-lung-cancer.

4. Recent literature suggests the presence of the bacterial genus Acidovorax particularly in lung cancer patients. In the manuscript, the authors did not provide details of the bacterial genera found. The author might consider comparing their findings with the paper and comment on why their microbiome could be different:

Greathouse, K.L., White, J.R., Vargas, A.J. et al. Interaction between the microbiome and TP53 in human lung cancer. Genome Biol 19, 123 (2018). https://doi.org/10.1186/s13059-018-1501-6.

5. Since the authors emphasize on the effect of bacteria on mortality, the author must include one paragraph on what has already been published on the possible role of microbial infections on mortality of cancer patients. Recent literature such as the following could have been cited.

Translational Oncology. VOLUME 14, ISSUE 12, P2097-2108, DECEMBER 01, 2019

Gram-Negative Pneumonia Augments Non–Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4. Stephen D. Gowing, Simon C. Chow, Jonathan J. Cools-Lartigue, Simon Rousseau, Salman T. Qureshi, Lorenzo E. Ferri, https://doi.org/10.1016/j.jtho.2019.07.023

Koslow M, Epstein Shochet G, Matveychuk A, Israeli-Shani L, Guber A, Shitrit D. The role of bacterial culture by bronchoscopy in patients with lung cancer: a prospective study. J Thorac Dis. 2017;9(12):5300-5305. doi:10.21037/jtd.2017.10.150

Ye M, Gu X, Han Y, Jin M, Ren T. Gram-negative bacteria facilitate tumor outgrowth and metastasis by promoting lipid synthesis in lung cancer patients. J Thorac Dis. 2016;8(8):1943-1955. doi:10.21037/jtd.2016.06.47

Kovaleva, O.V.; Romashin, D.; Zborovskaya, I.B.; Davydov, M.M.; Shogenov, M.S.; Gratchev, A. Human lung microbiome on the way to cancer. J. Immunol. Res. 2019, 2019, 1394191.

Chow, S. C. et al. Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation. Int J Cancer 136, 1341–1350, https://doi.org/10.1002/ijc.29111 (2015).

Minor corrections:

6. In Pg 3 line 56 cephalosporine spelling correction to Cephalosporin

7. In Pg 4 Line 83 says '(definition see below)' – no definition given

8. In Pg 4 Line 84 ‘…were included into this analysis…’ consider changing the word 'into' to 'to'.

9. In Pg 4 Line 85 ‘….second malignancy - aside of localized non-melanoma skin cancer….’ consider replacing ‘aside of’ by ‘aside from’

10. In Pg 5 Line 100 ‘…….unit as well as all patients all patients admitted to the thoracic surgery…..’, repetition of ‘all

patients’.

11. In Pg 14, Lines 289-290, ‘We encountered a significantly higher co-occurence of diabetes in patients’ spelling of occurrence.

Reviewer #2: I would like to congratulate the authors for conducting the study. It is a well written protocol. There are no previous studies on MDRO and lung cancer. The authors have clearly discussed the limitations.

Reviewer #3: The authors aimed to reveal that the impact of MDRO colonization in patients who have been diagnosed with Non-small cell lung cancer (NSCLC) who are at known high-risk for invasive infections. However, the data is not accurate enough. The data in this manuscript do not support the conclusion.

Reviewer #4: Authors have presented a very important topic on antimicrobial resistance in patients with NSCLC. Antibiotic resistance is a global problem which need be to tackled worldwide. Despite low prevalence of MDRO in their study, however this data from developed world merit to be shared to scientific community at large.

Overall the manuscript is well written and interesting to read. However, results and discussion sections need to revised. In results section some tables are difficult to understand and statistical interpretations are not well understood.

Specific comment

Abstract:

Line 38: Replace 295 with Two hundred and ninety-five.

Line 44: Add crude and adjusted, plus their 95%CI for both univariate and multivariable analysis, respectively.

Line 44 – 46: “There was a significantly higher rate of non-cancer-related mortality in MDROpos compared to MDROneg patients (p<0.001) with a trend MDROpos towards an increased rate of fatal infections in patients (p=0.05)” from table 3 I found very difficult to interpret. The way p-value which has been presented it looks to reject null hypothesis in favor of alternative hypothesis. Considering NRM cases alone in comparison of non-cancer related mortality might obscure the intended outcome, if possible include all participants in this analysis. Table 3 need to be revised for more clarity.

Introduction:

Line 55–56: Third-generation cephalosporin resistant should be replaced with extended spectrum beta lactamase producers.

Line 57: Replace Staphylococcus aureus with methicillin resistance to Methicillin resistance Staphylococcus aureus

Materials and Methods

ESBL, MRSA and VRE were screened by screening media, these media have high sensitivity. It will be interesting to state how this MDRO were confirmed either by phenotypic or genotypic.

Line 146: Ensure consistency either use MDROpos or MDRO+ .

Results

Line 168: Replace 271 with Two hundred and seventy-one.

Line 173: Diabetes mellitus did not show significant association, revisit supplementary file OR included 1.

Table1: Some variable in a column total % do not add to 100%. For example, in co-morbidity variable total number of MDRO is 25 and not 24. Interpretation along the row could be interesting than along the column. Think of revising this table.

Line 197 – 207: Specify name of specific antibiotics tested instead of using classes of antibiotics. For examples macrolide use either erythromycin or azithromycin, aminoglycosides specify gentamicin or others.

In analysis MDRO+ exposure to OS outcome. The disease stage could be one of the important confounders for OS. See Fig1A for example, for interest I would like to know if you controlled for disease stage as confounder, what happen HR. If you did not do please could you explain to me for interest.

Although this has been explained in discussion, however authors need to dig further on MDRO colonization with inferior overall survival outcome. It is well hypothesized MDRO colonization is a risk for severe MDRO infection with the same bacteria. MDRO infection could have direct effect on overall survival in these group of patients.

Analysis on Fig2B and 2C could be combined (IV/IIIB vs IIIA/II/I) rather than categorized in different group. Since disease staging in itself could predict OS.

Line 246 – 251 looks like figure 2 legend, please move below the figure2

Line 256 – 258: Check comment on abstract.

Table 3: Check comment on abstract

Line 267 – 268: Looks like legend for figure 3.

Discussion:

Well written, however in some part authors need to revise like line 302. If table 3 is revised this statement might need to change, there is a need to include all mortality not NRM only.

In conclusion, line 350 authors did not establish the correlation between MDRO colonization and infection. To state this as a reason “due higher rate of fatal infections mostly involving the lung tissue”. This infection was unrelated to colonization, the statement needs to be revised.

**********

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Reviewer #1: Yes: Professor Sunjukta Ahsan, Department of Microbiology, University of Dhaka, Bangladesh, sunjukta@du.ac.bd

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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13 Oct 2020

Dear Prof. Heber, Dear Richard Hodge

thank you for the possibility to re-submit our manuscript after major revision. We thank the referees for their valuable comments and have addressed all their concerns.

We hope the manuscript can now be accepted in Plos One and look forward to your response.

Sincerely,

Jan Stratmann, Sebastian Koschade

Comments to the referees:

Reviewer #1: The manuscript titled 'Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer' tries to show the importance of multi-drug resistant organisms on survival and mortality of patients suffering from non-small cell lung cancer. The main limitation of this investigation is the use of a very small sample to draw an important/significant conclusion.

Major comments:

1. The study sample size is small, only 24 out of 295 (8%) patients were studied. The sample size must be significantly larger than this to draw any valid conclusion.

We agree with the reviewer that the overall sample size is limited. Albeit conclusions derived from our work need confirmation in larger studies, we still believe our data is of importance for our readership, particularly due to increasing prevalence of MDRO worldwide and secondly due to the wide lack of clinical evidence regarding the impact of MDROs in oncological diseases.

We now acknowledge the limited sample size in the discussion section.

2. In Pg 2 Lines 42-46, the authors mention that 'There was a significantly higher rate of non-cancer-related-mortality in MDROpos patients compared to MDROneg patients (p<0.001) with a trend towards an increased rate of fatal infections in MDROpos patients (p=0.05). Also, Pg 11, Lines 231-232 states 'When stratified for disease stage (Fig. 2A), median OS in the MDROpos study group showed a significantly inferior median survival time in patients with advanced (IIIB) or metastatic disease (IV). Then again, in Pg 14, Line 302, the authors say ‘Approximately 80% of non-relapse mortality in the MDROpos group was infection-related.’

These are strong statements. Patients in advanced stages of cancer are more likely to suffer from mortality when compared to those in early stages of cancer simply because of their physiological conditions and not just because of bacterial colonization.

Bacterial infections in patients at early stage of cancer can actually augment progression to more critical stages/metastasis according to the existing literature. So, it is the severity of the infection at the early stage which determines or is one of the predisposing factors to progression to a more critical stage.

We agree with the reviewer and have revised the relevant sections. While suggestive, our retrospective trial design does not allow us to identify causal factors. In order to address this justified criticism, we mitigated our claims suggestive of causality throughout the manuscript. However, this does not pertain to our main finding of significantly poorer overall survival in MDROpos NSCLC patients (primary study outcome).

We agree with the notion that infection-related complications might also be a clinically relevant factor in relapsed/progressed patients and that an analysis of the distribution of infections between these patients might give additional information. However, we relied on the cause of death as described in the final medical report or on the death certificate. We unfortunately do not have sufficient information on the final course of each individual patient to judge the contribution of infectious-related complications to the death of patients with progressive cancer. However, we believe that information gained from this secondary/exploratory analysis is rather hypothesis generating than convincing evidence, we therefore – as mentioned above - weakened our claims regarding this analysis throughout the manuscript.

Regarding the impact on survival we have already adjusted for disease stage in our multivariate Cox proportional hazards regression analysis of overall survival (see Tab. 2, limited vs extensive disease as well as Fig. 2). As expected, extensive disease was highly significantly associated with a poorer OS (HR 3.03, 95% CI 2.16–4.24). The association between MDRO colonization and OS still remained significant in this multivariable analysis in the overall cohort (Tab. 2).

3. There are many existing literature that the authors can compare their work with such as:

i. Sarihan S, Ercan I, Saran A, Cetintas SK, Akalin H, Engin K. Evaluation of infections in non-small cell lung cancer patients treated with radiotherapy. Cancer Detect Prev. 2005;29(2):181-188. doi:10.1016/j.cdp.2004.11.001

ii. https://www.cancernetwork.com/view/infectious-complications-lung-cancer.

As we have pointed out in the introduction, infections are important complications in patients with lung cancer. We thank the reviewer for his suggestions on additional literature regarding (pulmonary) infections in these patients and have added more background data mainly to the introduction section.

4. Recent literature suggests the presence of the bacterial genus Acidovorax particularly in lung cancer patients. In the manuscript, the authors did not provide details of the bacterial genera found. The author might consider comparing their findings with the paper and comment on why their microbiome could be different:

Greathouse, K.L., White, J.R., Vargas, A.J. et al. Interaction between the microbiome and TP53 in human lung cancer. Genome Biol 19, 123 (2018). https://doi.org/10.1186/s13059-018-1501-6.

We highly appreciate the reviewer´s suggestion to compare the above-mentioned literature which is focusing on microbial characteristics / the bacterial consortium within the lung cancer (intratumoral) microenvironment. Contrary to Greathouse et al, we focused on the colonization with MDRO species that are detected during clinical routine. The microbial diversity either in the lung or gut in patients with specific cancer types is a competitive field of research at present, it is however not part of our research and data. Detailed data regarding the MDRO species in our study can be found in Table S4.

5. Since the authors emphasize on the effect of bacteria on mortality, the author must include one paragraph on what has already been published on the possible role of microbial infections on mortality of cancer patients. Recent literature such as the following could have been cited.

Translational Oncology. VOLUME 14, ISSUE 12, P2097-2108, DECEMBER 01, 2019

Gram-Negative Pneumonia Augments Non–Small Cell Lung Cancer Metastasis through Host Toll-like Receptor 4. Stephen D. Gowing, Simon C. Chow, Jonathan J. Cools-Lartigue, Simon Rousseau, Salman T. Qureshi, Lorenzo E. Ferri, https://doi.org/10.1016/j.jtho.2019.07.023

Koslow M, Epstein Shochet G, Matveychuk A, Israeli-Shani L, Guber A, Shitrit D. The role of bacterial culture by bronchoscopy in patients with lung cancer: a prospective study. J Thorac Dis. 2017;9(12):5300-5305. doi:10.21037/jtd.2017.10.150

Ye M, Gu X, Han Y, Jin M, Ren T. Gram-negative bacteria facilitate tumor outgrowth and metastasis by promoting lipid synthesis in lung cancer patients. J Thorac Dis. 2016;8(8):1943-1955. doi:10.21037/jtd.2016.06.47

Kovaleva, O.V.; Romashin, D.; Zborovskaya, I.B.; Davydov, M.M.; Shogenov, M.S.; Gratchev, A. Human lung microbiome on the way to cancer. J. Immunol. Res. 2019, 2019, 1394191.

Chow, S. C. et al. Gram negative bacteria increase non-small cell lung cancer metastasis via Toll-like receptor 4 activation and mitogen-activated protein kinase phosphorylation. Int J Cancer 136, 1341–1350, https://doi.org/10.1002/ijc.29111 (2015).

The suggested literature is of much interest for our data and we have put some of the above-mentioned publications into the context of our data.

Minor corrections:

6. In Pg 3 line 56 cephalosporine spelling correction to Cephalosporin

We have changed this accordingly.

7. In Pg 4 Line 83 says '(definition see below)' – no definition given

We have added a reference to the section “screening procedures and definitions” where the requested definitions can be found.

8. In Pg 4 Line 84 ‘…were included into this analysis…’ consider changing the word 'into' to 'to'.

This was corrected.

9. In Pg 4 Line 85 ‘….second malignancy - aside of localized non-melanoma skin cancer….’ consider replacing ‘aside of’ by ‘aside from’

We have changed this accordingly.

10. In Pg 5 Line 100 ‘…….unit as well as all patients all patients admitted to the thoracic surgery…..’, repetition of ‘all

patients’.

This was corrected.

11. In Pg 14, Lines 289-290, ‘We encountered a significantly higher co-occurence of diabetes in patients’ spelling of occurrence.

This was corrected.

Reviewer #2: I would like to congratulate the authors for conducting the study. It is a well written protocol. There are no previous studies on MDRO and lung cancer. The authors have clearly discussed the limitations.

Reviewer #3: The authors aimed to reveal that the impact of MDRO colonization in patients who have been diagnosed with Non-small cell lung cancer (NSCLC) who are at known high-risk for invasive infections. However, the data is not accurate enough. The data in this manuscript do not support the conclusion.

Reviewer #4: Authors have presented a very important topic on antimicrobial resistance in patients with NSCLC. Antibiotic resistance is a global problem which need be to tackled worldwide. Despite low prevalence of MDRO in their study, however this data from developed world merit to be shared to scientific community at large.

Overall the manuscript is well written and interesting to read. However, results and discussion sections need to revised. In results section some tables are difficult to understand and statistical interpretations are not well understood.

We thank the reviewer for the substantial time and effort and appreciate the kind comments and helpful suggestions.

Specific comment

Abstract:

Line 38: Replace 295 with Two hundred and ninety-five.

This has been replaced.

Line 44: Add crude and adjusted, plus their 95%CI for both univariate and multivariable analysis, respectively.

Line 43 and 44 report the Kaplan-Meier estimate of median overall survival (OS) in the MDROpos and MDROneg study groups with 95% CI. The Kaplan-Meier estimates of median survival itself or its associated CI is independent of covariables. We used multivariable Cox regression technique to adjust for additional, potentially confounding covariables in testing the association between MDRO colonization and OS (as expressed by hazard ratios and their CIs in Tab. 2). We report p values for the test of association between MDRO colonization and survival for both univariate and multivariate statistics. The multivariable Cox proportional hazards regression models would allow to estimate the probability of median survival in fixed subgroups of the data, e.g. for female MDROpos patients, or in the group of MDROpos and MDROneg patients while keeping all the other covariates fixed at their mean value. We are not aware that this is commonly done for the reporting of median survival times in trial populations.

Line 44 – 46: “There was a significantly higher rate of non-cancer-related mortality in MDROpos compared to MDROneg patients (p<0.001) with a trend MDROpos towards an increased rate of fatal infections in patients (p=0.05)” from table 3 I found very difficult to interpret. The way p-value which has been presented it looks to reject null hypothesis in favor of alternative hypothesis. Considering NRM cases alone in comparison of non-cancer related mortality might obscure the intended outcome, if possible include all participants in this analysis. Table 3 need to be revised for more clarity.

We agree with the reviewer that Table 3 is difficult to interpret. We have therefore revised Table 3 to display the data in a more intuitive way. The table legend has been rewritten to describe the statistical testing carried out in more detail in order to allow interpretation of the p value. Additionally, we have included a comment on the statistical test performed.

The null hypothesis being tested here was an equal distribution of infectious-related deaths in the MDROpos and MDROneg subgroups. We hypothesized that infectious-related deaths might account for the increased non-relapse mortality observed in MDROneg patients and therefore tested this. The death of the other participants was either due to relapse or progression or due to unknown/indeterminate causes. We agree with the notion that infection-related complications might also be a clinically relevant factor in relapsed/progressed patients and that an analysis of the distribution of infections between these patients might give additional information. However, we relied on the cause of death as described in the final medical report or on the death certificate. We unfortunately do not have sufficient information on the final course of each individual patient to judge the contribution of infectious-related complications to the death of patients with progressive cancer. However, we believe that information gained from this exploratory analysis is rather hypothesis generating than convincing evidence, we therefore weakened our claims regarding this analysis throughout the manuscript.

Introduction:

Line 55–56: Third-generation cephalosporin resistant should be replaced with extended spectrum beta lactamase producers.

We thank the reviewer for this suggestion. After careful consideration, we have opted not to make this change, since “third-generation cephalosporin resistant” also encompasses cephalosporine resistance which is not due to ESBLs but instead due to other cephalosporinases such as AmpC beta-lactamases in Enterobacter, Serratia and others (see e.g. DOI 10.1128/CMR.00036-08).

Line 57: Replace Staphylococcus aureus with methicillin resistance to Methicillin resistance Staphylococcus aureus

We have changed this accordingly.

Materials and Methods

ESBL, MRSA and VRE were screened by screening media, these media have high sensitivity. It will be interesting to state how this MDRO were confirmed either by phenotypic or genotypic.

As pointed out in the chapter “detection and molecular resistance analysis in MDRO”, VRE, ESBL and MRSA species are confirmed PHENOTYPICALLY by CLSI certified VITEK and/or agar diffusion method.

Line 146: Ensure consistency either use MDROpos or MDRO+ .

We have corrected this.

Results

Line 168: Replace 271 with Two hundred and seventy-one.

We have corrected this.

Line 173: Diabetes mellitus did not show significant association, revisit supplementary file OR included 1.

Table 1 shows that Diabetes mellitus was present in 50% of patients with MDRO colonization and 16% of MDROneg patients, and that this association was highly significant.

Table1: Some variable in a column total % do not add to 100%. For example, in co-morbidity variable total number of MDRO is 25 and not 24. Interpretation along the row could be interesting than along the column. Think of revising this table.

Comorbidites has non-mutually exclusive categories (some patients had multiple comorbidities), explaining while the patient numbers do not sum to 100%. However, while checking Table 1 we noticed that the entry “1st line treatment approach” was missing information for 5 patients. We have corrected this and apologize for the mistake.

Line 197 – 207: Specify name of specific antibiotics tested instead of using classes of antibiotics. For examples macrolide use either erythromycin or azithromycin, aminoglycosides specify gentamicin or others.

We have added this data.

In analysis MDRO+ exposure to OS outcome. The disease stage could be one of the important confounders for OS. See Fig1A for example, for interest I would like to know if you controlled for disease stage as confounder, what happen HR. If you did not do please could you explain to me for interest.

We agree with the reviewer that disease stage is a potential confounder for OS. We have therefore already adjusted for this in our multivariate Cox proportional hazards regression analysis of overall survival (see Tab. 2, limited vs extensive disease as well as Fig. 2). As expected, extensive disease was highly significantly associated with a poorer OS (HR 3.03, 95% CI 2.16–4.24). The association between MDRO colonization and OS remained significant in this multivariable analysis in the overall cohort (Tab. 2).

Although this has been explained in discussion, however authors need to dig further on MDRO colonization with inferior overall survival outcome. It is well hypothesized MDRO colonization is a risk for severe MDRO infection with the same bacteria. MDRO infection could have direct effect on overall survival in these group of patients.

We agree with the reviewer that MDRO colonization is a likely risk factor for infection by the colonizing bacteria. In support of this hypothesis, we have therefore statistically tested the rate of mortality due to infections in MDROpos vs. MDROneg patients and observed statistical significance (p=0.002) towards an increased rate of infection in MDROpos patients (see revised Table 3; this relates to the reviewer’s remark and our answer above in regard to Table 3.). At the end of the results section (‘Cause of death’), we further discuss all MRDOpos patients within our cohort which died due to complications relating to infections and also provide information regarding the colonizing pathogens later detected in invasive infections, as well as discuss the infections.

Analysis on Fig2B and 2C could be combined (IV/IIIB vs IIIA/II/I) rather than categorized in different group. Since disease staging in itself could predict OS.

We agree with the reviewer that disease stage should be part of the full survival analysis, since it likely impacts survival. As outlined in our answer above, we have already included the disease stage as a variable in the multivariable survival analysis. We have included the separate (IV/IIIB vs IIIA/II/I) Kaplan-Meier OS estimates in Fig. 2B and 2C because we believe it is of clinical interest to depict the associative impact of MDRO colonization in these two stages. However, our primary outcome analysis (multivariable survival analysis) is performed with the full patient cohort, taking into account the disease stage for each patient. The association between MDRO colonization and OS remained significant in the multivariable analysis of the overall cohort (HR 1.96, 95% CI 1.09-3.51; see Tab. 2).

Line 246 – 251 looks like figure 2 legend, please move below the figure2

We totally agree with the reviewer that placing the Figure´s legends into the running text (and not under the corresponding Figure) is confusing, unfortunately this is part of the PlosOne author guidelines, so we are not able to place the legend(s) below the corresponding figures.

Line 256 – 258: Check comment on abstract.

Table 3: Check comment on abstract

As outlined in our answer above, we have revised Table 3 and our discussion of it.

Line 267 – 268: Looks like legend for figure 3.

Please see above.

Discussion:

Well written, however in some part authors need to revise like line 302. If table 3 is revised this statement might need to change, there is a need to include all mortality not NRM only.

In conclusion, line 350 authors did not establish the correlation between MDRO colonization and infection. To state this as a reason “due higher rate of fatal infections mostly involving the lung tissue”. This infection was unrelated to colonization, the statement needs to be revised.

We agree with the reviewer and have revised this section. No claim of causality is made any more in this regard. While suggestive, our retrospective trial design does not allow us to identify causal factors. In order to address this justified criticism, we mitigated our claims suggestive of causality throughout the manuscript. However, this does not pertain to our main finding of significantly poorer overall survival in MDROpos NSCLC patients.

Submitted filename: 200920 Response to Reviewers.docx

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3 Nov 2020

PONE-D-20-09321R1

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PLOS ONE

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PLOS ONE

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Reviewers' comments:

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: (No Response)

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However, the author needs to correct one spelling mistake, that is 'thirdly' in line 326. Otherwise, the manuscript is acceptable.

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Reviewer #1: Yes: Dr. Sunjukta Ahsan, Department of Microbiology, University of Dhaka, Bangladesh

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3 Nov 2020

Reviewer #1: The authors have considered all suggestions and corrections addressed by the reviewer. Comments have been added and additional references have been cited as suggested. Minor corrections have also been addressed. The most outstanding limitations were acknowledged. This makes the manuscript more acceptable from a readers point of view.

However, the author needs to correct one spelling mistake, that is 'thirdly' in line 326. Otherwise, the manuscript is acceptable.

We thank the referee again for his time to review our revised manuscript and have applied the last suggested change.

Reviewer #2: The authors have answered all queries. I have no further comments, The manuscript will be useful for the readers.

We thank the reviewer for his valuable time.

Submitted filename: 201103 Response to Reviewers.docx

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5 Nov 2020

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

PONE-D-20-09321R2

Dear Dr. Stratmann,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Joel Manyahi

Guest Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

12 Nov 2020

PONE-D-20-09321R2

Colonization with multi-drug-resistant organisms negatively impacts survival in patients with non-small cell lung cancer

Dear Dr. Stratmann:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Guest Editor

PLOS ONE