The importance of active surveillance of carbapenem-resistant Enterobacterales (CRE) in colonization rates in critically ill patients.

OBJECTIVE: This study aimed to demonstrate the importance of active carbapenem-resistant Enterobacterales (CRE) surveillance and evaluate the prevalence of invasive infections, risk factors, and mortality risk in CRE-colonized patients.
METHODS: Retrospective cohort study analyzing 1,920 patients identified using an active CRE surveillance protocol, admitted to an adult intensive care unit in southeastern Brazil from January 2014 to December 2018.
RESULTS: There were 297 (15.47%) CRE colonized patients, with one colonized for every six control patients. CRE-colonized patients demonstrated an increased chance of infection (odds ratio [OR] 7.967, p < 0.001). Overall, 20.54% of the colonized patients presented invasive infection (81.96% due to Klebsiella pneumoniae). The colonization and infection ratio demonstrated the important role of the active CRE surveillance protocol. There were identified multiple risk factors for CRE colonization, including long-term mechanical ventilation (OR 1.624, p = 0.019) and previous exposure to aminopenicillins (OR 5.204, p < 0.001), carbapenems (OR 3.703, p = 0.017), cephalosporins (OR 12.036, p < 0.001), and fluoroquinolones (OR 5.238, p = 0.012). The mortality risk was significantly higher among colonized (OR 2.356, p < 0.001) and colonized-infected (OR 2.000, p = 0.009) patients and in those with Enterobacter cloacae colonization (OR 5.173, p < 0,001) and previous aminopenicillins exposure (OR 3.452, p = 0.007).
CONCLUSIONS: Early detection of CRE colonization through screening testing proved to be an important tool to control CRE spread. However, observation over the years has shown no effective control of colonization and infection. The prevalence rates of CRE colonization and colonization-infection were high, as were the mortality rates. In conclusion, an active CRE surveillance protocol is essential, but its impact depends on the effective implementation of preventive measures and feedback between team members.

Introduction

Colonization by carbapenem-resistant Enterobacterales (CRE) is an important cause of infection and one of the main sources of CRE dissemination in hospitals and in the community [1,2].

Dissemination of CRE has been occurring at an alarming pace worldwide due to the low response of these bacteria to available therapies and their difficult management with empirical antibiotics [3,4].

Consequently, there has been a high prevalence of CRE colonization and infection with serious threats to public health [5-10].

In asymptomatic carriers, the main CRE reservoir is the microbiota in the gastrointestinal tract, followed by the oropharynx, skin, and urine [11,12]. These colonization reservoirs are considered essential in the CRE spread inside and outside the hospital environment [11,13-16].

The genes determining Enterobacterales antimicrobial resistance have emerged in the past two decades [2,7,9] due to selective pressure caused by the wide use of broad-spectrum antibiotics, especially in intensive care units (ICUs) and long-stay hospitals [1,6,8,11,17,18].

Health-care units adopting specific antibiotic dispensing rules for each type of infection have individual microbial biosystems [13,17]. However, these treatments are not optimized and lead to increased resistance rates, including resistance to third generation cephalosporins (10–97%) [13,17]. This highlights the relevance of multidrug-resistance (MDR) bacteria in healthcare-associated infections (HAIs) [8,13,19-22].

Colonization by CRE can be acquired directly after antibiotic exposure or due to cross-transmission between patients and healthcare professionals [2,14]. The patients most susceptible to CRE colonization are those who are severely ill, have comorbidities, are subjected to invasive devices, and have previous antibiotic exposure, and long-term hospitalization [8,9,22,23].

Active CRE surveillance testing is an important strategy to control the spread of MDR bacteria, as it allows early implementation of protective measures through contact isolation, positively impacting public health [4,9,23-25]. The United States Centers for Disease Control and Prevention (CDC) considers the detection of CRE through rectal swabs the preferable method of CRE screening [25].

Patients with CRE colonization have a high probability of developing a subsequent infection that may be associated with bacteremia, leading to increased morbidity and mortality (30–75%) [7,8,10,17,24,26-28].

The occurrence of HAI caused by CRE is a serious matter since these infections frequently fail to respond to the delivered therapy, resulting in long-term hospital stay and increased hospital costs [10,13-15].

According to current CDC data, the mortality associated with antibiotic-resistant infections has decreased in the United States between 2013 and 2019 [29]. However, the number of registered infections caused by resistant bacteria remains high, with over 2.8 million cases/year and 35,000 deaths/year [29].

Based on these considerations, the aim of this study was to demonstrate the importance of active CRE surveillance in the ICU and verify the prevalence of CRE invasive infections in CRE-colonized and control patients. The study also aimed to determine the risk factors and risk of mortality related to strains, previous antibiotic exposure, and type of CRE colonization.

Methods

Study design

This was a retrospective cohort study including CRE-colonized patients, carried out in a 30 beds adult ICU of a public tertiary hospital in southeastern Brazil.

The study included patients older than 12 years, hospitalized in an adult ICU from January 2014 to December 2018. The patients were subjected to the active surveillance protocol for CRE detection and prevention established by the Hospital’s Infection Control Commission (CCIH) in April 2011 (09/2011 Minutes). The resolution follows the guidelines from the Brazilian National Health Surveillance Agency (ANVISA) [30] and the criteria for antimicrobial resistance established by the World Health Organization (WHO) [31] and the CDC [25]. The active CRE surveillance protocol determines measures for detection with screening testing using rectal swabs collected upon the patient’s admission and at weekly intervals during hospitalization, while the prevention measures are executed only for the cases confirmed of CRE colonization or infection. They include strict contact isolation in a properly identified with precaution contact signs private room, rapid and safe patient discharge, restricted use of probes and catheters, protection and hygiene of healthcare professionals, cleaning of the environment, and rational dispensing of antibiotics.

The research protocol was approved by the institution’s Research Ethics Committee (protocol number 1.638.131).

Data collection

A total of 2,126 active CRE surveillance protocol records were collected from 2012 to 2018. After review, patients’ records who were discharged or died before the first rectal swab collection, as well as those who had incomplete or duplicated records and the period from 2012 to 2013 due to irregularities in the collections were excluded. After a final review of the protocol records, 1,920 patients subjected to the CRE surveillance protocol from January 2014 to December 2018 were included in the analysis. Demographic, clinical, and microbiological data from each individual record were collected, including the patients’ identification record, age, sex, clinical diagnosis upon admission, disease severity score according to the Simplified Acute Physiology Score (SAPS) II, use of invasive devices, identification of Enterobacterales and other microorganisms, use of antimicrobials, results of rectal swabs for detection of CRE, and dates of admission and discharge or death.

The patients’ records provided information regarding the length of ICU stay, use of long-term (over 10 days) mechanical ventilation, use of long-term (over 15 days) hemodialysis catheters, antibiotic exposure 30 days before the colonization and/or infection, and late death (30 days after admission). Some of the variables were stratified according to observed averages such as younger (13–54 years) and older (55–97 years) age groups, low (16–62) or high (63–217) disease severity scores (SAPS II), and short-term (1–21 days) or long-term (22–175 days) ICU stay.

The infected patients were clinically diagnosed with CRE infection and tested positive for CRE cultures, according to infection guidelines by the CDC’s National Healthcare Safety Network (NHSN) [32]. The cultures of clinical and surveillance samples were subjected to identification and susceptibility tests using the VITEK2 automated system (BioMérieux, France) according to the Clinical and Laboratory Standard Institute (CLSI) guidelines [33]. The turnaround time mean of the CRE surveillance results was of five days. In case of positive clinical or surveillance culture for CRE, the microbiology laboratory informs immediately the medical or nursing staff of the ICU and the CCIH makes notifications in the medical electronic record and prescription.

Statistical analysis

The clinical data are presented as frequencies. Continuous variables with normal distribution are presented as mean and standard deviation values and were compared using Student’s t test. The normality of the distribution of quantitative variables was analyzed using the Shapiro-Wilk test. Pearson’s chi-square test or Fisher’s exact test was used to analyze the samples of colonized and non-colonized patients for infection and to determine the risk factors for colonization. The risk factors that were statistically relevant (p < 0.05) were submitted to a multivariate logistic regression test to determine the effect of all risk factors on the colonized and control patients, and to a multinomial logistic regression test to determine the influence of factors on CRE-colonized and colonized-infected patients in relation to controls. Multiple Cox regression, the Kaplan-Meier method, and the log-rank test were used to estimate survival by comparing curves between colonized, colonized-infected, and control groups. Multiple Cox regression was also used to calculate the proportional hazards of types CRE colonizers and antibiotic exposure previous to colonization. The adopted significance level (α) for all analyses was below 5% (p- value < 0.05). The strength of the association between the explanatory variables and the response was assessed by calculating the odds ratio (OR) with 95% confidence intervals (95% CIs). The analyses were carried out using the statistical software SPSS for Windows (IBM Corp., Armonk, NY, USA).

Results

Among all 1,920 patients, the mean age was 52.42 ± 19.34 years (range 13–97 years), and there was a predominance of the male (65.31%) over the female (1.88:1) sex. Discharge (68.12%) was a more predominant outcome than death (2.13:1). The SAPS II score ranged from 16 to 131 (mean 62.19 ± 18.73). The mean length of hospital stay was 21.03 ±18.12 days (range 1–175 days).

A total of 3,154 rectal swabs were collected, of which 2,807 were negative for CRE in 1,623 patients (range 1–15 swabs/patient, mean 1.74 swabs/patient) and 344 were positive (10.91%) in 297 patients (range 1–3 positive swabs/patient, mean 1.16 swabs/patient). The most frequent strains identified were Klebsiella pneumoniae (83.16%), followed by Enterobacter cloacae (9.76%), Escherichia coli (4.38%), Enterobacter aerogenes (1.34%), Serratia marcescens (0.67%), Enterobacter gergoviae (0.34%), and Serratia fonticola (0.34%). Three patients had two rectal swabs each that isolated different CRE microorganisms; the first swab identified Klebsiella pneumoniae (in all three patients) and the second identified Enterobacter cloacae (in two patients) and Serratia fonticola (in one patients).

The patients were categorized into two groups: (1) colonized patients (n = 297, 15.47%), comprised by patients who tested positive in the CRE surveillance testing (including colonized-infected patients) and (2) control patients (n = 1,623, 84.53%), comprised by patients without CRE colonization (i.e., negative surveillance testing). In the colonized and control patient groups, there was predominance of the male (66.32% and 65.12%, respectively) over the female sex (1.97:1 and 1.87:1, respectively) and more discharges (67.34% and 68.26%, respectively) in relation to deaths (2.06:1 and 2.15:1, respectively). The CRE-colonized group differed significantly from the control group in terms of SAPS II score (range 16–127, mean 65.54 ± 17.96 and range 16–131, mean 61.57 ± 18.80, respectively, p = 0.01), and length of hospital stay (interval 3–175 days, mean 33.40 ± 24.47 days and interval 1–145 days, mean 18.77 ± 15.68 days, respectively, p < 0.001) but not regarding age (range 14–97 years, mean 53.85 ± 19.37 years and range 13–93 years, mean 52.16 ± 19.33 years, respectively, p = 0.167).

Invasive CRE infections were detected in 61 (20.54%) of the 297 CRE-colonized patients and in 51 (3.14%) of the 1,623 control patients. Among the colonized-infected patients, the following CRE strains predominated in different collection sites: K. pneumoniae (81.96%) in urine, blood, tracheobronchial secretion, and tissue secretion; E. cloacae (16.39%) in blood, central venous catheter tip, and tissue secretion; and E. coli (4.92%) in urine and blood. Rates of other CRE strains were not significantly different in colonized-infected patients compared with controls, i.e., S. marcescens (1.64%) in urine, E. aerogenes (1.64%) in blood, and E. gergoviae (1.72%) in tracheobronchial secretion. Clinical infections caused by two strains in different cultures were identified in four patients colonized by K. pneumoniae; these included two patients with K. pneumoniae and E. cloacae, one patient with K. pneumoniae and E. coli, and one patient with K. pneumoniae and E. aerogenes. One patient colonized with E. coli was identified as having an invasive infection by E. coli and E. cloacae.

The infection to colonization ratio across the duration of the study were 1:3.5 in 2014, 1:2.4 in 2015, 1:3.6 in 2016, 1:8.3 in 2017, and 1:7.2 in 2018. Fig 1 shows the infected to colonized ratio for each year of the study.

Fig 1

Analysis of infection rates prior to colonization and of total colonized and total infected rates.

Clinical invasive infections by the same colonizing CRE microorganisms occurred in 53 (17.84%) of the 297 colonized patients, of which 48 (90.56%) were K. pneumoniae. Analysis of CRE-colonized patients demonstrated an increased chance of infection with these strains (Table 1).

Table 1

Univariate analysis of the risk of development of invasive clinical CRE infection in the sample.

Infection	CRE colonized n (%)	Controls n (%)	OR (95% CI)	p
Yes	61 (20.54)	51 (3.14)	7.967 (5.361–11.841)	<0.001
No	236 (79.46)	1572 (96.86)
Total	297	1623

CRE = Carbapenem-resistant Enterobacterales, n = number of patients, OR = odds ratio, 95% CI = confidence interval, p = test significance.

Risk factors for colonization were identified by assessing the effect of all independent variables that showed statistically significant differences (p < 0,05) in the comparative analysis of colonized and control patients (Table 2).

Table 2

Univariate analysis and multivariate logistic regression analysis of risk factors for CRE colonization in colonized versus non-colonized patients.

Variables	CRE colonized n (%)	Non-colonized n (%)	Bivariate		Multivariate
Total no. of patients	N = 297 (15.47)	N = 1623 (84.53)	OR (95% CI)	p	OR (95% CI)	p
Age range (55 to 97 Years)	159 (53.54)	804 (49.54)	1.174 (0.916–1.504)	0.208	-	-
Sex (male)	197 (66.33)	1057 (65.13)	0.948 (0.730–1.231)	0.740	-	-
SAPS (63 to 217)	172 (57.91)	759 (46.77)	1.566 (1.220–2.012)	0.001	0.733 (0.558–0.965)	0.027
ICU stays (22 to 175 days)	175 (58.92)	500 (30.81)	3.222 (2.499–4.154)	<0.001	1.099 (0.762–1.585)	0.614
Clinical diagnosis at ICU admission	n (%)	n (%)	OR (95% CI)	p	OR (95% CI)	p
Septic shock	26 (8.75)	134 (8.26)	1.066 (0.687–1.655)	0.733	-	-
Acute respiratory distress	30 (10.10)	123 (7.58)	1.370 (0.900–2.085)	0.161	-	-
Neoplasm	22 (7.41)	99 (6.10)	1.232 (0.762–1.989)	0.366	-	-
Postoperative	26 (8.75)	150 (9.24)	0.942 (0.609–1.457)	0.913	-	-
Lower level of consciousness	43 (14.48)	270 (16.64)	0.848 (0.599–1.202)	0.393	-	-
Infectious syndrome	16 (5.39)	51 (3.14)	1.755 (0.987–3.121)	0.059	-	-
Inflammatory syndrome	18 (6.06)	95 (5.85)	1.038 (0.617–1.745)	0.893	-	-
Metabolic syndrome	20 (6.73)	77 (4.74)	1.450 (0.872–2.410)	0.151	-	-
Trauma	96 (32.32)	624 (38.45)	0.765 (0.588–0.955)	0.050	-	-
Invasive devices	n (%)	n (%)	OR (95% CI)	p	OR (95% CI)	p
Central venous catheter	290 (97.64)	1474 (90.82)	4.188 (1.942–9.030)	<0.001	1.972 (0.863–4.507)	0.107
Long-term hemodialysis catheter	41 (13.80)	91 (5.61)	2,696 (1.822–3.989)	<0.001	1.225 (0.779–1.928)	0.379
Tracheostomy	210 (70.71)	638 (39.31)	3.727 (2.848–4.877)	<0.001	0.617 (0.406–0.939)	0.024
Long-term MV	218 (73.40)	710 (43.75)	3.548 (2.694–4.674)	<0.001	1.624 (1.085–2.430)	0.019
Long-term urethral catheter	282 (94.95)	1439 (88.66)	2.404 (1.399–4.131)	0.001	1.186 (0.646–2.177)	0.583
Enteral feeding tube	268 (90.24)	1266 (78.00)	2.606 (1.746–3.891)	<0.001	1.315 (0.848–2.038)	0.221
Long-term gastric tube	38 (12.79)	115 (7.09)	1.924 (1.303–2.841)	0.002	1.023 (0.664–1.575)	0.919
Previous antibiotic exposure	n (%)	n (%)	OR (95% CI)	p	OR (95% CI)	p
Aminopenicillins	14 (4.71)	10 (0.62)	7.980 (3.210–18.141)	<0.001	5.204 (2.244–12.066)	<0.001
Carbapenems	7 (2.36)	7 (0.43)	5.572 (1.940–16.005)	0.003	3.703 (1.259–10.893)	0.017
Cephalosporins	38 (12.79)	14 (0.86)	16.862 (9.011–31.555)	<0.001	12.036 (6.225–23.271)	<0.001
Fluoroquinolones	6 (2.02)	4 (0.25)	8.345 (2.341–29.755)	0.002	5.238 (1.443–19.009)	0.012

CRE = Carbapenem-resistant Enterobacterales, n = number of patients, OR = odds ratio, 95% CI = confidence interval; p = test significance, SAPS = Simplified Acute Physiology Score, MV = mechanical ventilation, ICU = intensive care unit.

Following that, the association of these risk factors for colonization was analyzed by separately comparing the colonized and colonized-infected groups in relation to the control group. This final multivariable model showed also significant predictors for each group (Table 3).

Table 3

Multinomial regression analysis of risk factors for patients with CRE colonization and colonization-infection compared with controls.

Variables	Colonized (n = 236)			Colonized-infected (n = 61)
	OR	95% CI	p	OR	95% CI	p
SAPS (63 to 217)	1.433	1.070–1.921	0.016	1.127	0.638–1.990	0.681
Central venous catheter	2.061	0.859–4.946	0.105	1.467	0.175–12.296	0.724
Long-term hemodialysis catheter	0.980	0.588–1.632	0.937	2.490	1.206–5.141	0.014
Tracheostomy	1.472	0.935–2.316	0.095	2.703	0.976–7.489	0.056
Long-term MV	1.329	0.864–2.044	0.196	6.731	2.008–22.556	0.002
Long-term urethral catheter	1.067	0.576–1.975	0.836	2.756	0.448–31.467	0.222
Enteral feeding tube	1.348	0.847–2.148	0.208	1.098	0.364–3.313	0.868
Long-term gastric tube	0.873	0.536–1.421	0.585	1.621	0.797–3.296	0.182
Aminopenicillins	4.513	1.820–11.189	0.001	8.745	2.473–30.918	0.001
Carbapenems	2.565	0.728–9.034	0.143	9.223	2.125–40.037	0.003
Cephalosporins	8.363	4.085–17.122	<0.001	35.021	14.224–86.222	<0.001
Fluoroquinolones	3.230	0.708–14.742	0.130	15.114	3.012–75.842	0.001
Long- term ICU stay	1.365	0.914–2.038	0.128	0.447	0.209–0.955	0.038

CRE = Carbapenem-resistant Enterobacterales, n = number of patients, OR = odds ratio, 95% CI = confidence interval; p = test significance, SAPS = Simplified Acute Physiology Score, MV = mechanical ventilation, ICU = intensive care unit, n controls = 1623.

On multinomial logistic regression, there was a predominance of CRE strains in invasive devices of colonized-infected patients. The strains included K. pneumoniae in long-term hemodialysis catheters (OR 2.387, 95% CI 1.187–4.799, p = 0.015), tracheotomy (OR 3.262, 95% CI 1.093–9.733, p = 0.034), and long-term mechanical ventilation (OR 5.931, 95% CI 1.484–23.705, p = 0.012), as well as E. cloacae in long-term hemodialysis catheters (OR 4.624, 95% CI 1.274–16.782, p = 0.020).

Multiple Cox regression analysis was performed to evaluate the survival rate of patients with CRE the colonization by K. pneumoniae (OR 2.206, 95% CI 1.468–3.316, p < 0.001), E. cloacae (OR 5.173, 95% CI 2.372–11.281, p < 0.001), and other CRE (E. aerogenes, E. coli, S. marcescens, E. gergoviae, and S. fonticola) in relation to control patients. The other CRE bacteria were grouped since their individual analysis was not statistically significant (OR 1.398, 95% CI 0.640–3.057, p = 0.401) (Fig 2).

Fig 2

Multiple Cox regression analysis of the estimated survival in patients with CRE-colonized bacteria compared with controls.

Multiple Cox regression analysis was also performed to evalute the mortality risk in CRE-colonized patients with previous antibiotic exposure (Fig 3). The mortality of patients who were colonized 30 days after use of antibiotics showed significant differences for the classes of aminopenicillins (OR 3.452, 95% CI 1.398–8.523, p = 0.007) and other grouped antibiotic classes such as carbapenems, cephalosporins, and fluoroquinolones (OR 1.829, 95% CI 1.078–3.102, p = 0.025), compared with the control group.

Fig 3

Multiple Cox regression analysis of the risk estimation for previous antibiotics exposure in CRE-colonized patients compared with controls.

Survival probability analysis showed higher mortality in colonized-infected and colonized patients compared with controls (log-rank test p < 0.001) (Fig 4). Multiple Cox regression analysis of the variable “death” after 30 days of ICU stay among CRE-colonized patients showed a high mortality risk with significant differences in colonized (OR 2.356, 95% CI 1.547–3.587, p < 0.001) and colonized-infected (OR 2.000, 95% CI 1.187–3.368, p = 0.009) patients compared with controls.

Fig 4

Kaplan-Meier curves of estimated survival of colonized and colonized-infected patients compared with controls.

Discussion

Patients colonized by MDR bacteria are considered to be important reservoirs since they favor horizontal transmission of these microorganisms in the hospital environment [6,16].

Healthcare without safety standards for hand hygiene can amplify the risk of spreading resistant strains in hospital environments and threaten human health with an increased risk of infection-related morbidity and mortality [34].

In addition to the responsibility of healthcare teams, limited resources for CRE surveillance screening in underdeveloped countries is an additional concern for implementation measures for the detection of resistant bacteria since these delays the implementation measures for infection control and prevention [35,36]. There is still no consensus in the literature about the best method to detect CRE colonization, even though rectal swabs have been shown to be a sensitive method and with good correlation in the process of screening for active surveillance [15]. Based on that, rectal swabs are considered the main screening method for active surveillance [25].

In the literature, the positivity rate of CRE screening testing using rectal swab is about 10.1%, and the most frequent strains identified are K. pneumoniae (7.9‒98.7%), followed by E. cloacae (22.0%), E. coli (20.0‒82.1%), and other CRE (5.0%) [3,4,12,22,36-38]. In the present study, CRE screening testing carried out using rectal swab had a positivity rate of 10.91%, and the predominant strains were K. pneumoniae (84.17%), E. cloacae (9.76%), and E. coli (4.38%).

Multifaceted programs seeking control of intra-hospital CRE transmission have frequently targeted intervention measures by healthcare teams [39]. The literature has shown CRE colonization rates of 8.8% to 18.9% in patients in long-term units and 28% in transplant units [12,16,22,36]. The present study showed a high colonization rate (15.47%) in the sample, identifying at least one colonized patient for every six patients in the ICU.

The role of prevention of CRE infection and colonization goes beyond the identification of carriers through surveillance testing, as it also depends on continuous efforts to achieve multidisciplinary adherence to protective isolation, hand hygiene, environmental cleaning, and appropriate antibiotic dispensation [2,25].

In 2016, a study on safety culture analysis was conducted in the same ICU of the present study to evaluate the multidisciplinary team using the Safety Attitudes Questionnaire (SAQ) [40]. This safety culture study demonstrated reliable and significant results regarding safety perception but observed weak attitudes regarding management perception, working conditions, and communication failures [40]. The present study observed relevant data regarding colonization and infection rates and demonstrated the importance of active CRE surveillance for the implementation of early preventive measures. Even with these measures, the infection and colonization rates in the ICU were high during the study period.

Critically ill CRE-colonized patients are more prone to develop invasive infections with broad resistance to available antibiotics [28]. However, the disease’s severity varies according to the pathogen’s virulence and the host’s potential defense and exposure to medical procedures [12,22,41]. According to the literature, CRE-colonized patients have an increased odd of developing infection (OR 2.06, p = 0.040), with rates ranging from 7.6 to 86.4% [3,7,10,12,26,27]. The infections developed by CRE-colonized patients have been predominantly by K. pneumoniae, followed by E. coli, K. oxytoca and E. cloacae [3,4,6,9,12,16]. In the present study, clinical CRE infection occurred in 20.54% of the CRE-colonized patients, corresponding to odds of infection of 7.967 (p < 0.001). K. pneumoniae, predominated in urinary, bloodstream, pulmonary, and subcutaneous tissue infections, while E. cloacae predominated in blood and pulmonary infections.

The factors that increase the risk of a critical patient becoming susceptible to CRE colonization are several and included tracheostomy (OR 4.8, p < 0.001), enteral feeding tube (OR 3.3, p = 0,001), long-term hospital stay (OR 3.8, p = 0.045), previous carbapenem exposure (OR 2.54, p < 0.05), and invasive procedures (OR 2.18, p < 0.05) [4,6,9,12,22,25]. The present study also revealed multiple risk factors for CRE colonization, including long-term mechanical ventilation (OR 1.624, p = 0.019) and previous exposure to aminopenicillins (OR 5.204, p < 0.001), carbapenems (OR 3.703, p = 0.017), cephalosporins (OR 12.036, p < 0.001), and fluoroquinolones (OR 5.238, p = 0.012).

Colonization by CRE is a pathogenic condition considered to be a strong determinant for the development of infection [3,6,22,26]. Some risk factors act as infection facilitators in colonized patients, including previous use of antibiotics such as fluoroquinolones (OR 3.04, p = 0.037), mechanical ventilation, non-surgical invasive medical procedures (OR 2.18, p < 0.05), endoscopy or colonoscopy (OR 3.7, p = 0.02), previous hospitalizations, and long-term ICU stay (OR 7.45, p = 0.023) [3,6,12,22,26,42]. In the present study, the risk factors for colonization and infection were similar but more prevalence in CRE colonized-infected patients compared with colonized and control patients. The risk factors in colonized-infected patients include long-term use of hemodialysis catheters (OR 2.490, p = 0.014), long-term mechanical ventilation (OR 6.731, p = 0.002), and previous exposure to aminopenicillins (OR 8.745, p = 0.001), carbapenems (OR 9.223, p = 0.003), cephalosporins (OR 35.021, p < 0.001), and fluoroquinolones (OR 15.114, p = 0.001).

The identification of modifiable risk factors and antibacterial resistance profile in hospital units brings positive results to protective measures, with a consequent reduction mortality [6,7,12]. Among the modifiable risk factors, stood out in the present study the predominance of infection by K. pneumoniae in invasive devices (81.96% in colonized-infected patients), including long-term mechanical ventilation, tracheotomy, and long-term hemodialysis catheters, and by E. cloacae (16.39% in colonized-infected patients) in long-term hemodialysis catheters. The risk of mortality was significantly higher in patients colonized-infected by E. cloacae compared with other CRE.

Antibiotic dispensing programs determine the class of the antibiotic to be used, optimizing the antibiotic dose and limiting its duration of use [21,43]. These measures are helpful in HAI therapeutic responses and reducing the selection of drug-resistant microorganisms [44]. The importance of this appropriate control can be seen in the results of the present study, in which the mortality risk was significantly higher in patients with previous use of aminopenicillins and other antibiotic classes such as carbapenems, cephalosporins, fluoroquinolones, and polymyxins.

The most important adverse outcomes in CRE-colonized patients include infection and mortality, the latter with rates in the literature ranging from 27.5% to 41% [3,12,18]. The present study observed higher mortality in colonized-infected patients, followed by colonized ones.

Conclusion

Early detection of CRE colonization through active CRE surveillance testing demonstrated to be important for implementing the necessary measures to contain the spread of these multiresistant microorganisms. However, observation over the years showed an absence of effective control of both CRE colonization and infection. The CRE-colonized patients had increased odds of infection, while colonized-infected patients had a higher risk of mortality. The occurrence of CRE colonization and colonization-infection presented known risk factors that can be modified through daily surveillance of signs of infection in catheters, daily awakening of the patients to disconnect mechanical ventilation, early removal of catheters and tubes, and implementation of antibiotic dispensing programs. In short, CRE active surveillance testing is essential, but its impact depends on effective actions in implementing preventive measures and feedback between team members.

6 Oct 2021

PONE-D-21-27215The importance of active surveillance of carbapenem-resistant Enterobacteriaceae (CRE) in colonization rates in critically ill patientsPLOS ONE

Dear Dr. Gomides,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please provide clarifications on the points raised by the reviewers and correct the discrepancy in the dates mentioned in different sections of the manuscript.

Please submit your revised manuscript by Nov 20 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Journal Requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please include in your Methods section the full name of the participating hospitals/institution.

3. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Two reviewers have commented on the manuscript and raised some very important questions and clarifications. Please address all comments point by point.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: 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 Aims and objectives to be clearly defined

Line 94 and 138- enterobacteria is it a standard terminology it must be Enterobacteriaceae

Invasive infections were found be more in colonized patients than the control group ,is it after application of all the preventive measures as per the hospital policy.

Line 228 and 231- to be written as association of risk factors and colonization not influence needs statistical consultation for using these terminologies

Reviewer #2: The manuscript highlights an important problem of gut colonization with CRE in a high risk population and I wish the best to the authors.

Please consider the following review comments :

1) Carbapenem resistant Enterobacteriaceae (CRE) should be replaced with "CR Enterobacterales" at all places including the title

2) Pg 5 : study design , line 115 : The method of processing the rectal swab must be elaborated for the benefit of the readers along with a reference

3) Pg 6 : data collection , line 130 mentions data collection from 2012 to 2018. Then Line 133: mention data included from 2014 to 2018. Please mention clearly the duration of the study

4) No clarity on the following issues :

• Wheter patients were kept in contact isolation till results arrived?

• What was the turn around time of the CRE surveillance results?

• What media were used for surveillance purposes?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Aruna Poojary

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

19 Oct 2021

In response to the reviewers’ comments to the Author

Reviewer #1: The Aims and objectives to be clearly defined

Line 94 and 138- enterobacteria is it a standard terminology it must be Enterobacteriaceae

Answer Line 94: I did not identify the enterobacteria word at line 94.

Answer Line 138: enterobacteria was replaced by “Enterobacteriaceae”.

Invasive infections were found be more in colonized patients than the control group, is it after application of all the preventive measures as per the hospital policy.

Answer: Yes, invasive infections in colonized patients occurred after application of all the preventive measures.

Line 228 and 231- to be written as association of risk factors and colonization not influence needs statistical consultation for using these terminologies

Answer: It’s correct! The terminology was replaced after consult the statistical books.

Reviewer #2: The manuscript highlights an important problem of gut colonization with CRE in a high risk population and I wish the best to the authors.

Answer: Thanks!

Correction the following review comments:

1) Carbapenem resistant Enterobacteriaceae (CRE) should be replaced with "CR Enterobacterales" at all places including the title

Answer: "CR Enterobacterales" was replaced at all places including the title

2) Pg 5: study design, line 115: The method of processing the rectal swab must be elaborated for the benefit of the readers along with a reference

Answer pg 5: study design, line 115: The method of processing the rectal swab was written of the clear form on page 6: data collection, line 151, along with a reference.

3) Pg 6: data collection, line 130 mentions data collection from 2012 to 2018. Then Line 133: mention data included from 2014 to 2018. Please mention clearly the duration of the study

Answer pg 6: data collection, line 130 and 133: The duration of the study was mentioned for best comprehension of the readers.

4) No clarity on the following issues:

• Whether patients were kept in contact isolation till results arrived?

Answer: No, the patients weren’t kept in contact isolation till the results arrived. Therefore, the phrase of the pg. 5: study design, line 122 was reformulated for best comprehension the readers.

• What was the turnaround time of the CRE surveillance results?

Answer: The turnaround time mean of the CRE surveillance results was of five days. These data were described on pg. 7: data collection, line 154, for best comprehension of the readers.

• What media were used for surveillance purposes?

Answer: The media used for surveillance purposes were information immediate of the microbiology laboratory and notifications in the electronic record and prescription medical by Hospital Infection Control Sector. These data were described on pg. 7: data collection, line 155, for best comprehension of the readers.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

22 Nov 2021

PONE-D-21-27215R1The importance of active surveillance of carbapenem-resistant Enterobacterales (CRE) in colonization rates in critically ill patientsPLOS ONE

Dear Dr. Gomides,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Two minor comments from Reviewer 2 to be addressed.

==============================

Please submit your revised manuscript by Jan 06 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Please see two minor comments from Reviewer 2.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

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

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

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

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 the comments are addressed and incorporated in the manuscript. The study is an important research work adds to the medical literature the relevance of looking for multi drug resistant bacterial colonizers and implement preventive measures to control their spread in the critical areas of the health settings

Reviewer #2: Thank you for clarifying the comments. Pls find below the comments from the fresh review

1) Pg 66 , line 73 : Capital E for Enterobacterales

2) Pg 69 line 141: pls change to Enterobacterales

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: Aruna Poojary

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

24 Dec 2021

Rebuttal Letter

In response to the reviewers’ comments to the Author

Items 1 to 5 - compliance between reviewers and article

________________________________________

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 the comments are addressed and incorporated in the manuscript. The study is an important research work adds to the medical literature the relevance of looking for multi drug resistant bacterial colonizers and implement preventive measures to control their spread in the critical areas of the health settings

Answer: Thanks your comments!

Reviewer #2: Thank you for clarifying the comments. Pls find below the comments from the fresh review

1) Pg 66, line 73: Capital E for Enterobacterales

Answer Line 73: enterobacterales was replaced by “Enterobacterales”.

2) Pg 69 line 141: pls change to Enterobacterales

Answer Line 138: Enterobacteriaceae was replaced by “Enterobacterales”.

________________________________________

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Answer: Thanks your review in this article!

Reviewer #2: Yes: Aruna Poojary

Answer: Thanks your review in this article!

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Dec 2021

The importance of active surveillance of carbapenem-resistant Enterobacterales (CRE) in colonization rates in critically ill patients

PONE-D-21-27215R2

Dear Dr. Gomides,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Iddya Karunasagar

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

All reviewer comments have been addressed.

Reviewers' comments:

7 Jan 2022

PONE-D-21-27215R2

The importance of active surveillance of carbapenem-resistant Enterobacterales (CRE) in colonization rates in critically ill patients

Dear Dr. Gomides:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Iddya Karunasagar

Academic Editor

PLOS ONE