Risk Factors for Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Carriage in Patients Admitted to Intensive Care Unit in a Tertiary Care Hospital in Thailand

Abstract

Extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) are important causes of serious infections in intensive care unit (ICU). This study aimed to investigate the risk factors for intestinal carriage of ESBL-PE among patients admitted to ICU, subsequent ESBL-PE infections, and outcomes of these patients. This study prospectively collected rectal swabs from 215 ICU patients in Northern Thailand and ESBL-PE were isolated. A high prevalence of ESBL-PE carriage (134/215, 62.3%) at ICU admission was observed, with Escherichia coli representing the predominant organism (67.5%) followed by Klebsiella pneumoniae (19.4%). Multivariate logistic regression analysis identified chronic renal disease as the independent risk factor for ESBL-PE carriage (p = 0.009; adjusted odds ratio = 4.369; 95% confidence interval = 1.455–13.119). Among colonized patients, 2.2% (3/134) developed ESBL-PE infections during ICU stay. Phylogenetic analysis of E. coli (n = 108) showed that the predominant group was group A (38.0%), followed by groups B1 (17.6%), D (15.7%), B2 (14.8%), C (7.4%), and F (6.5%). Multilocus sequence typing analysis of the pathogenic groups B2, D, and F revealed 11 different sequence types (STs), with ST131 (n = 13) as the most prevalent, followed by ST648 (n = 5), ST38 (n = 4), ST393 (n = 3), and ST1193 (n = 3). These results are of concern since ESBL-PE may be a prerequisite for endogenous infections and potentially disseminate within the hospital. This is the first study describing ESBL-PE carriage among patients at ICU admission and subsequent ESBL-PE infections in Thailand.

Introduction

Multidrug-resistant (MDR) Gram-negative bacteria cause life-threatening infections in hospital settings, particularly carbapenem-resistant Enterobacteriaceae (CRE), which are considered a serious threat to health care facilities and have now spread to several countries. However, in Southeast Asia, the prevalence of CRE remains infrequent.¹ A recent report from the National Antimicrobial Resistance Surveillance Thailand (NARST) involving 69 hospitals during January 2018–June 2018 revealed that ∼2.8% and 12.8% of Escherichia coli and Klebsiella pneumoniae, respectively, were resistant to carbapenems.² In contrast, infections caused by extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-PE) are comparatively high and ESBL producers, instead of CRE, are considered to be a major health issue in Southeast Asian region.¹ In a recent study, Thailand was listed as the second highest of 13 countries in the Asia-Pacific region for intra-abdominal infections caused by ESBL-PE.³

ESBL-PE have long contributed to hospital- and community-associated infections in different parts of Thailand.^4,5 Nosocomial infections caused by ESBL-PE are highly prevalent⁶ and the clonal spread of ESBL-PE within the hospital has previously been reported.⁷ Furthermore, Thai patients infected with ESBL-PE resulted in poor clinical outcomes and high mortality rates.⁴

A link between prior intestinal carriage of ESBL-PE and subsequent colonization and infection had been demonstrated.^8,9 Furthermore, many studies have shown that surveillance of ESBL-PE colonization at hospital admission prevented the spread of ESBL-PE or infection with ESBL-PE within the hospital.^10,11 Among hospitalized patients, those admitted to intensive care unit (ICU) are considered to be at high risks of developing serious infections leading to an increase in morbidity and mortality, hospitalization, and health care costs. A recent study reported that ICU patients colonized with ESBL-PE have ∼50 times higher risk in developing ESBL-PE infections compared with the noncolonized patients.¹² The colonized ESBL-PE isolates may serve as reservoirs for developing endogenous infections especially in immunocompromised patients and may spread to other patients. Alternatively, intestinal flora may develop resistance as a result of plasmid transfer from the colonizing-resistant bacteria.¹³ Therefore, a better knowledge of ESBL-PE colonization in patients admitted to ICU may be useful for implementing appropriate infection control measures and, thereby, limiting ESBL-PE spread within hospitals. Furthermore, data on ESBL-PE colonization may help clinicians in guiding appropriate antimicrobial treatment as a means to limit carbapenem usage and perhaps, reducing the emergence of CRE.

Studies from different countries demonstrated the varying ESBL-PE carriage rates among patients admitted to ICUs. These are usually low in the United States and Europe (<15%)^14–16 but are repeatedly reported to be high in Asian countries such as India and Vietnam.^17,18 However, to our knowledge, data from Thailand are not available. In this study, we aimed to investigate the prevalence and risk factors for intestinal colonization of ESBL-PE among patients admitted to ICU in a tertiary care hospital in Northern Thailand and to examine the number of colonized patients who had subsequent ESBL-PE infections as well as the outcome of these patients.

Materials and Methods

Study design, setting, and patients

This prospective cohort study was conducted in ICU of Buddhachinaraj Hospital, a 1,000-bed tertiary care hospital in Phitsanulok province, Northern Thailand, between December 2014 to November 2015. All adult patients ≥20 years of age with no intestinal tract infection were enrolled in this study. In case of readmission of the same patient during the period of study, only the first admission was studied.

A total of 215 ICU patients were enrolled in this study. Rectal swabs (Amies swab; Deltalab, Barcelona, Spain), obtained within 48 hr of ICU admission, were collected from each patient. Demographic characteristics, previous antibiotic usage, and hospitalization from each patient were collected by a structured questionnaire. Data on underlying conditions and current diseases, length of ICU stay, nosocomial infections, and outcome of patients were obtained from patients' medical records.

The present study was approved by the Naresuan University Institutional Review Board (COA No. 130/2013). Written informed consent was obtained patients' next of kin before participating in the study.

Microbiological studies

Rectal swab samples were directly cultured on Chrome UTI agar (Oxoid, Basingstoke, United Kingdom) supplemented with vancomycin (25 μg/mL) and cefotaxime (1 μg/mL) and incubated under aerobic condition for 24 hr at 37°C. Colonies presenting different morphologies were selected and subcultured onto Tryptic Soy Agar for further studies. Species identification was performed by biochemical tests using RapID™ONE System (REMEL, Inc., KS). For isolates that showed unreliable results, 16S rDNA sequencing, using primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (1,502 bp), was used to identify bacterial species.¹⁹ Screening for ESBL production was performed by combination disc method according to Clinical Laboratory Standards Institute (CLSI) protocols using cefotaxime and ceftazidime alone or in combination with clavulanic acid.²⁰ Patients were considered to be colonized if one or more ESBL-producing organisms was recovered from each rectal swab. The prevalence of ESBL-PE carriage was calculated as the percentage of carriers among participants of each group.

All isolates were subjected to susceptibility testing against 13 antimicrobial agents (cefotaxime, ceftazidime, cefepime, aztreonam, imipenem, meropenem, ertapenem, ciprofloxacin, levofloxacin, gentamicin, amikacin, doxycycline, and trimethoprim–sulfamethoxazole) by the disc diffusion method according to CLSI protocols.²⁰ An isolate was considered to be MDR if it was nonsusceptible to at least one agent in three or more antimicrobial categories.²¹

Detection of genes encoding for CTX-M and carbapenemase by PCR and sequencing

The presence of genes encoding for CTX-M and carbapenemase was screened by multiplex PCR using previously published primers and conditions.^22,23 Individual colony was suspended in 1 mL of water and was used as template in PCR. Amplification was performed in a total volume of 20 μL containing 1 μL of template DNA, 1 × PCR buffer, 1.5 mM MgCl₂, 0.2 mM dNTPs, and 0.5 μM of each primer and 1 U of Taq polymerase (Vivantis Technologies, Selangor, Malaysia). Selected PCR products were purified using a DNA Purification Kit (GF-1 Nucleic Acid Extraction Kit; Vivantis, Inc., Chino, CA) and sequenced. Nucleotide sequences were compared with those available in the GenBank database using the BLAST algorithm available on the National Center for Biotechnology Information (NCBI) website (www.ncbi.nlm.nih.gov).

Identification of bla_CTX-M alleles was performed by PCR using primers: bla_{CTX-M group 1}, 5′-ATGGTTAAAAAATCACTGCG-3′, and 5′-TTACAAACCGTCGGTGAC-3′ (876 bp); bla_{CTX-M group 8}, 5′-TCGCGTTAAGCGGATGATGC-3′ and 5′-TTAATAACCGTCGGTGACG-3′ (864 bp); and bla_{CTX-M group 9}, 5′-CAAAGAGAGTGCAACGGATG-3′ and 5′-TTACAGCCCTTCGGCGATGA-3′ (866 bp). The PCR conditions were 5 min of initial denaturation at 94°C, followed by 30 cycles at 94°C for 1 min, 48°C (52°C for bla_{CTX-M group 8}) for 45 sec, and 72°C for 1 min and a final extension at 72 at 94°C for 5 min. Nucleotide sequence analysis was performed as described above.

Phylogenetic grouping and multilocus sequence typing analysis

Phylogenetic grouping (A, B1, B2, C, D, E, and F) of E. coli was performed by a multiplex PCR assay as previously described.²⁴ Multilocus sequence typing (MLST) analysis of pathogenic groups B2, D, and F was performed by amplification and sequencing of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) according to the protocols from E. coli MLST website (http://enterobase.warwick.ac.uk/species/ecoli/allele_st_search).

Statistical analyses

All statistical analyses were performed using SPSS version 17.0 (SPSS, Chicago, IL). Proportions were compared using z-test. The differences were considered statistically significant at p < 0.05. Categorical variables were shown as numbers with percentages and continuous variables were presented as mean or median with ranges. Initial univariate analyses were performed by Chi-square test or Fischer's exact test, as appropriate, for categorical variables, and Mann–Whitney U test for continuous variables. Variables with p-value <0.1 in univariate analyses were further analyzed in multivariate logistic regression analysis using a backward stepwise procedure to determine risk factors associated with intestinal carriage of ESBL-PE. The results were presented as adjusted odds ratios (aORs) with 95% confidence interval (CI). All tests were two-tailed and a p < 0.05 was considered a significant difference.

Results

Intestinal carriage of ESBL-producing Enterobacteriaceae

Two hundred and fifteen ICU patients were enrolled in this study and their clinical information is listed in Table 1. The male/female ratio was 1.1 (113/102) and the mean and median ages were 60.8 and 62 (range, 20–97) years, respectively. The main reason for admission was respiratory tract disease. The most common underlying condition was cardiovascular diseases, followed by diabetes. Overall, 62.3% (134/215, 95% CI = 55.5–68.8) of patients were colonized with ESBL-PE. One hundred and nine patients (109/215, 50.7%) carried a single species of ESBL-PE. Carriage of at least two ESBL-PE species was found in 25 patients (25/215, 11.6%). Among these, 24 and 1 possessed two and three ESBL-PE isolates, respectively. Prevalence rate of patients who were colonized with a single species of ESBL producer was significantly higher than that colonized with two or more isolates of ESBL producers (50.7% vs. 11.6%, p < 0.001).

Table 1.

Clinical Characteristics for Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Carriage Among Patients Admitted to Intensive Care Units (n = 215)

Risk factors	Total no. of patients	ESBL-PE carrier (n = 134)	non-ESBL-PE carrier	Univariate analysis p-value	Multivariate logistic regression
	Total no. of patients	ESBL-PE carrier (n = 134)	non-ESBL-PE carrier		p-Value^a	Adjusted odd ratio	95% CI
	(n = 215) n (%)	n (%)	(n = 81) n (%)		p-Value^a	Adjusted odd ratio	95% CI
Demographic data
Male gender	113 (52.6)	66 (49.3)	47 (58.0)	0.212
Mean age	60.8	61.4	59.9	0.419
Farmer	71 (33.0)	44 (32.8)	27 (33.3)	0.970
Secondary school education	151 (70.2)	94 (70.1)	57 (70.4)	0.949
No. of family members (>5 persons)	42 (19.5)	23 (17.2)	19 (23.5)	0.258
Family income (<10,000 Baht)^b	138 (64.2)	85 (63.4)	53 (65.4)	0.783
Urban living	49 (22.8)	29 (21.6)	20 (24.7)	0.701
Antibiotic usage within previous 3 months	74 (34.4)	48 (35.8)	26 (32.1)	0.592
Hospitalization within previous 6 months	66 (30.7)	45 (33.6)	21 (25.9)	0.251
Origin of patients
Home	49 (22.8)	35 (26.1)	14 (17.3)	0.107
Another hospital	68 (31.6)	43 (32.1)	25 (30.9)	0.743
Another ward within hospital	89 (41.4)	49 (36.6)	40 (49.4)	0.09	0.289	0.724	0.399–1.314
Principle diagnosis
Respiratory tract disease	59 (27.4)	39 (29.1)	20 (24.7)	0.403
Cardiovascular disease	31 (14.4)	20 (14.9)	11 (13.6)	0.719
Sepsis	33 (15.3)	24 (17.9)	9 (11.1)	0.153
Brain disease	56 (26.0)	29 (21.6)	27 (33.3)	0.078	0.107	0.592	0.313–1.119
Underlying conditions
Cardiovascular disease	108 (50.2)	69 (51.5)	39 (48.1)	0.486
Diabetes	55 (25.6)	36 (26.9)	19 (23.5)	0.496
Respiratory tract disease	30 (14.0)	16 (11.9)	14 (17.3)	0.314
Renal disease	29 (13.5)	25 (18.7)	4 (4.9)	0.003	0.009	4.369	1.455–13.119
Liver disease	11 (5.1)	6 (4.5)	5 (6.2)	0.621

p-Value <0.05 was considered statistically significant.

Average minimum income/month = 10,000 Baht.

ESBL-PE, extended-spectrum β-lactamase-producing Enterobacteriaceae; CI, confidence interval.

Overall, 160 isolates of ESBL-PE were recovered. E. coli was the predominant species (108/160, 67.5%), followed by K. pneumoniae (31/160, 19.4%) and Enterobacter cloacae (6/160, 3.8%). Other ESBL-PE included Klebsiella spp, Enterobacter aerogenes, Kluyvera spp., Serratia spp., Salmonella spp., and Shigella spp. (Table 2).

Table 2.

Distribution of Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae Isolates and Their bla_CTX-M

	No. (%) of isolates carrying bla_CTX-M
ESBL-PE (n, %)(n = 160)	bla_{CTX-M-group 1}		bla_{CTX-M-group 8}	bla_{CTX-M-group 9}				bla_{CTX-M-group 1} + bla_{CTX-M-group 9}			Total
ESBL-PE (n, %)(n = 160)	bla_CTX-M-15	bla_CTX-M-55	bla_CTX-M-63	bla_CTX-M-9	bla_CTX-M-14	bla_CTX-M-27	bla_CTX-M-65	bla_CTX-M-14,-15	bla_CTX-M-14,-55	bla_CTX-M-15,-27	Total
Escherichia coli (n = 108, 67.5)	20	25			23	11	1	7	9	1	97
Klebsiella pneumoniae (n = 31, 19.4)	14	1	4								19
Enterobacter cloacae (n = 6, 3.8)	1			1		1					3
Klebsiella oxytoca (n = 3, 1.9)			1								1
Citrobacter spp. (n = 3, 1.9)	1	1			1						3
Enterobacter aerogenes (n = 2, 1.3)					1						1
Klebsiella spp. (n = 1, 0.6)	1										1
Other Enterobacteriaceae^a (n = 6, 3.8)				1	2						3
Total (n = 160)	37 (23.1)	27 (16.9)	5 (3.1)	2 (1.3)	27 (16.9)	12 (7.5)	1 (0.6)	7 (4.4)	9 (5.6)	1 (0.6)	128 (80.0)

Other Enterobacteriaceae isolates include Kluyvera spp. (n = 2), Serratia spp. (n = 2), Salmonella spp. (n = 1), and Shigella spp. (n = 1).

Susceptibility testing

Susceptibility testing was performed and the results showed that all isolates were uniformly resistant to cefotaxime. Almost all isolates were resistant to cefepime (156/160, 97.5%), ceftazidime (150/160, 93.8%), and aztreonam (148/160, 92.5%). The majority of isolates were resistant to ciprofloxacin (125/160, 78.1%), doxycycline (124/160, 77.5%), trimethoprim/sulfamethoxazole (101/160, 63.1%), gentamicin (101/160, 63.1%), amikacin (93/160, 58.1%), and levofloxacin (82/160, 51.3%). In addition, 20% (32/160), 7.5% (12/160), and 6.9% (11/160) of isolates were resistant to imipenem, ertapenem, and meropenem, respectively. Furthermore, 156 isolates (156/160, 97.5%) were classified as MDR.

Detection of genes encoding for CTX-M and carbapenemase

Of the 160 ESBL-PE isolates, 128 isolates (128/160, 80%) carried bla_CTX-M, including bla_{CTX-M group 1}, bla_{CTX-M group 8}, and bla_{CTX-M group 9}. Identification of bla_CTX-M alleles revealed that the most common bla_{CTX-M-group 1} was bla_CTX-M-15 (37/160, 23.1%,) followed by bla_CTX-M-55 (27/160, 16.9%). bla_CTX-M-14 was predominant (27/160, 16.9%) among bla_{CTX-M-group 9}-carrying isolates. In addition, bla_CTX-M-27 (12/160, 7.5%), bla_CTX-M-9 (2/160, 1.3%), and bla_CTX-M-65 (1/160, 0.6%) were detected at low frequencies. bla_CTX-M-63, belonging to the bla_{CTX-M- group 8}, was found in five isolates (5/160, 3.1%). Interestingly, 17 isolates (17/160, 11.9%), from 17 patients, were positive for both bla_{CTX-M group 1} and bla_{CTX-M group 9}. That is, bla_CTX-M-14,-15 (7/160, 4.4%), bla_CTX-M-14,-55 (9/160, 5.6%), and bla_CTX-M-15,-27 (1/160, 0.6%) (Table 2). Carbapenemase gene (bla_IMP, bla_VIM, bla_NDM, bla_KPC, and bla_OXA-48) was not found in any of the carbapenem-resistant isolates.

Risk factors, subsequent infections, and outcomes of ESBL-PE colonized patients

No significant differences in terms of demographic characteristics (age, gender, occupation, education, number of household family member, family income, place of living) and previous antibiotic usage between ESBL-PE carriers and noncarriers were observed (Table 1). Univariate analysis revealed that the only risk factor associated with ESBL-PE colonization among patients at ICU admission was chronic renal disease (p = 0.003), which was confirmed in multivariate logistic regression analysis (p = 0.009; aOR = 4.369; 95% CI = 1.455–13.119) (Table 1). The mean and median length of ICU stay for the studied patients were 8.5 and 6 (range, 1–34) days, respectively. We found that the length of ICU stay and nosocomial infections were similar for both ESBL-PE carriers and noncarriers (Table 3).

Table 3.

Outcome of Patients Colonized with Extended-Spectrum β-Lactamase-Producing Enterobacteriaceae at Intensive Care Unit Admission (n = 215)

Variables	No. of ESBL-PE carriers (n = 134), n (%)	No. of non-ESBL-PE carriers (n = 81), n (%)	p
Mortality	24 (17.9)	8 (9.9)	0.118
Nosocomial infection	16 (11.9)	9 (11.1)	1.000
Length of stay (day), (mean ± SD)	2–34 (8.6 ± 7.01)	1–28 (8.1 ± 5.9)
<8 days	80 (59.7)	47 (58.0)	0.886
8–14 days	25 (18.7)	21 (25.9)	0.232
15–21 days	13 (9.7)	9 (11.1)	0.818
>21 days	8 (6.0)	3 (3.7)	0.541

Among the 134 ESBL-PE colonized patients, 3 patients (2.2%) developed nosocomial infections due to ESBL-PE, including urinary tract infections (n = 2) and pulmonary infection (n = 1). Two patients who developed nosocomial ESBL-PE infections died due to sepsis and pneumonia after staying in ICU for 5 and 26 days, respectively.

In addition, nine colonized patients developed ICU-acquired infections caused by non-ESBL-PE, including Enterobacter spp. (n = 1), K. pneumoniae (n = 1), Proteus mirabilis (n = 1), Staphylococcus spp. (n = 1), Pseudomonas aeruginosa (n = 2), Streptococcus agalactiae (n = 1), vancomycin-resistant Enterococcus faecium (n = 1), and Acinetobacter baumannii (n = 1). Four colonized patients had positive cultures for multiple bacteria such as A. baumannii and E. faecium (n = 1), A. baumannii and MRSA (n = 1), as well as Streptococcus and Staphyloccoccus spp. (n = 2).

Phylogenetic grouping and MLST analysis of E. coli

Phylogenetic grouping was investigated for 108 E. coli isolates. The results showed that the predominant group was group A (41/108, 38.0%), followed by group B1 (19/108, 17.6%), D (17/108, 15.7%), B2 (16/108, 14.8%), C (8/108, 7.4%), and F (7/108, 6.5%). Phylogroup E was not identified. MLST analysis was performed on E. coli belonging to pathogenic groups B2, D, and F. Among 16 isolates of B2 group, 13 and 3 isolates were identified as ST131 and ST1193, respectively. The carriage rate of E. coli B2-ST131 among ICU patients was 6.0% (13/215). Seven isolates of E. coli group F were assigned as ST648 (n = 5), ST117, and ST1340 (one isolate each). Of the 17 isolates belonging to group D, 11 isolates were identified as ST38 (n = 4), ST393 (n = 3), ST349, ST405, ST1722, and ST3045 (one isolate each), whereas 6 isolates were untypeable. The distribution of the bla_CTX-M for E. coli isolates belonging to pathogenic groups B2, D, and F is shown in Table 4. These isolates carried either bla_CTX-M-14, bla_CTX-M-15, bla_CTX-M-27, bla_CTX-M-55, or a combination of two bla_CTX-M.

Table 4.

Distribution of bla_CTX-M Among Escherichia coli Pathogenic Groups B2, D, and F

Group/ST	No. of isolates	bla_CTX-M
B2/131	13	bla_CTX-M-15 (n = 6)
		bla_CTX-M-27 (n = 5)
		bla_CTX-M-14 (n = 2)
B2/1193	3	bla_CTX-M-27 (n = 2)
B2/1193	3	bla_CTX-M-55 (n = 1)
D/1722	1	bla_CTX-M-55 (n = 1)
D/3045	1	bla_CTX-M-14 + bla_CTX-M-15 (n = 1)
D/349	1	bla_CTX-M-55 (n = 1)
D/38	4	bla_CTX-M-14 (n = 1)
		bla_CTX-M-15 (n = 1)
		bla_CTX-M-27 (n = 2)
D/393	3	bla_CTX-M-27 (n = 1)
		bla_CTX-M-55 (n = 1)
		bla_CTX-M-15 + bla_CTX-M-27 (n = 1)
D/405	1	bla_CTX-M-15 (n = 1)
F/648	5	bla_CTX-M-14 (n = 3)
		bla_CTX-M-14 + bla_CTX-M-15 (n = 1)
		bla_CTX-M-14 + bla_CTX-M-55 (n = 1)
F/117	1	bla_CTX-M-15 (n = 1)
F/1340	1	bla_CTX-M-14 + bla_CTX-M-55 (n = 1)

ST, sequence type.

Discussion

Despite the controversy as to whether the active surveillance for ESBL-PE colonization among patients at hospital admission is useful for infection control measures, a recent report showed that screening of ESBL-PE carriers improved health outcomes and reduced costs for ESBL-PE infections.²⁵ The recent meta-analysis based on 10 studies involving 6,199 patients showed that the pooled prevalence of ESBL-PE colonization among patients at ICU admission was 10%.¹² However, in our study, we found a higher prevalence (62.3%) of ESBL-PE carriage among patients at ICU admission. This value was much higher than those reported from Asian countries such as Japan (13.6%)²⁶ and South Korea (28.2%),²⁷ but was comparable to that found in our neighbouring country, Vietnam (52.3%).¹⁸ It is not surprising as ESBL-PE fecal carriage in the community in Southeast Asian countries, particularly Thailand, has been shown to be strikingly high.²⁸ In addition, the high prevalence of ESBL-PE carriage found in our study was consistent with the carriage rates of ESBL-PE among healthy subjects (58.6–69.3%) in the same area and other parts of Thailand.^29,30 The high carriage rate may be linked to the poor living conditions and inappropriate use of antibiotics in Thailand.^28,31 It is noted that 11.6% of patients were colonized by multiple ESBL-PE isolates (≥2 species), which might increase the risk of developing hospital-acquired infections or might be more likely to have had treatment failure, if infection develops.

Chronic renal disease was identified as an independent risk factor for ESBL-PE colonization and this result could be explained by the fact that individuals who have chronic renal disease frequently visit hospital for dialysis. Accordingly, they may have increased contact with inanimate objects or environments contaminated with ESBL-PE. In addition, several studies have shown that chronic renal disease is frequently associated with ESBL-PE carriers or CRE carriers, although it was not identified as a risk factor.^8,32

Microbiological data revealed that ESBL-producing E. coli was the most frequent organism in the intestinal tract of patients (67.5%) compared with other Enterobacteriaceae species, in contrast to the data from Africa where ESBL-producing K. pneumoniae was significantly predominant among colonized patients.³³ These differences may, to some extent, reflect the epidemiology of ESBL-PE between regions. bla_CTX-M-15 was the most common ESBL-encoding gene followed by bla_CTX-M-55 and bla_CTX-M-14 (Table 2), consistent with the frequent ESBL genes reported in both community and hospital-acquired infections worldwide.³⁴ The high occurrence of bla_CTX-M is worrisome since ESBL-PE may transfer ESBL-gene to other normal flora within the intestinal tract, which had been demonstrated in a patient with cystic fibrosis.³⁵ ESBL-PE isolates with multiple bla_CTX-M (bla_CTX-M-14,-15, bla_CTX-M-14,-55, and bla_CTX-M-15,-27) were not desirable since they may confer resistance to various antimicrobial classes compared with isolates carrying a single ESBL gene.³⁶ Thirty-two ESBL-PE isolates were negative for bla_CTX-M, suggesting that other ESBL genes such as TEM- and SHV-related ESBL may be involved.³⁴

ESBL-PE are frequently associated with resistance to other classes of antibiotic families leading to the emergence of MDR organisms,³⁴ consistent with our results, which revealed that 97.5% of isolates were considered to be MDR. Therefore, initial empirical antibiotics for ICU patients colonized with ESBL-PE should be carefully selected to provide patients with favorable clinical outcomes. No carbapenemase-encoding gene (bla_IMP, bla_VIM bla_NDM bla_KPC, and bla_OXA-48) was found in our study despite the fact that 20% of isolates showed resistance to imipenem. These results were similar to those previously reported in human volunteers.³⁰ Further experiments are underway to investigate the mechanism of carbapenem resistance in these isolates.

Subsequent infections with ESBL-PE among colonized patients were not common. We observed that only three colonized patients developed ESBL-PE infections during ICU stay. The low incidence of ICU-acquired ESBL-PE infections found in this study may result from the compliance with infection control measures, including antibiotic stewardship and contact precautions, in our hospital. No differences regarding length of ICU stay were seen between carriers and noncarriers; however, we noted that the mortality rate of ESBL-PE carriers (17.9%) is higher than that of noncarriers (9.9%) although this is not statistically different (p = 0.118, Table 3).

E. coli species can be divided into seven main phylogroups namely A, B1, B2, C, D, E, and F. Of these, groups B2, D, E, and F are considered as extraintestinal pathogenic E. coli (ExPEC).²⁴ Our study revealed that the majority of isolates were commensal groups A and B1. However, we found that 40 isolates were classified in the ExPEC groups B2 (n = 16), D (n = 17), and F (n = 7), which were further analyzed by MLST. Of ExPEC isolates, E. coli ST131 is an international clone that typically possesses a wide variety of virulence factors, causes a variety of infections, and is strongly associated with resistance to fluoroquinolone and extended-spectrum cephalosporins.³⁷ In this study, we found that 13 ICU patients carried E. coli B2/ST131, harboring either bla_CTX-M-15, bla_CTX-M-27, or bla_CTX-M-14, in their intestinal tract (Table 4). These results are of concern since intestinal colonization by ST131 has been shown to be significantly longer than that of other E. coli lineages,³⁸ which may have profound effects on critically ill patients. Furthermore, E. coli ST131 has been shown to be 3.2 times more pathogenic than non-ST131³⁹ and has recently been reported to be frequently associated with hospital-acquired pneumonia in Thailand.⁴⁰

While ST131 is the predominant strain causing serious infections globally, E. coli ST1193, ST648, ST393, ST405, and ST38 are human ExPEC isolates that have been described in various hospital- and community-acquired infections, such as bacteremia and urinary tract infections, in several countries, including Thailand.^41–43 E. coli ST1193, in particular, is emerging as a fluoroquinolone-resistant variant of ExPEC clonal group and is considered as the second most common pathogenic group B2, after ST131.⁴⁴ Although, the clinical significance and epidemiology of these isolates in Thailand remain unclear, the presence of bla_CTX-M-positive E. coli ST1193, ST648, ST393, ST405, and ST38 in the intestinal tract of ICU patients is worrisome since these isolates are considered to be MDR organisms and may represent another cause of serious extraintestinal infections.

Limitations of this study include (1) the ESBL-PE strains that caused nosocomial infections in three colonized patients were not available for analysis, thus we did not know whether or not patients developed infections from the colonized strains and (2) the number of participating patients was relatively small, which may diminish the strength of risk factor analysis. Nevertheless, our findings have added to the growing data on the epidemiology of ESBL-PE and should raise awareness of infection control issues among hospital staffs in Thailand.

In conclusion, this is the first study describing a high intestinal colonization of ESBL-PE among patients at admission to ICU in Thailand and chronic renal disease is the only independent risk factor associated with this colonization. Our study further demonstrated that subsequent infections with ESBL-PE among colonized patients was rare despite the high colonization rate. Of particular interest, we identified E. coli ST131, which may be a prerequisite for extraintestinal infections in colonized patients, and be able to disseminate within the hospital.

Footnotes

Acknowledgments

This work was supported by Naresuan University (R2558B042). The authors are grateful to the nursing staffs at Infection Control Unit and ICU at Buddhachinaraj Hospital, Phitsanulok, Thailand for their help in collection of rectal swab samples and medical data. Anong Kiddee was financially supported by the Royal Golden Jubilee PhD program from Thailand Research Fund and Naresuan University (PHD/0181/2557).

Disclosure Statement

No competing financial interests exist.

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