Antimicrobial resistance and epidemiology of extended spectrum-β-lactamases (ESBL)-producing Escherichia coli and Enterobacter cloacae isolates from intensive care units at obstetrics & gynaecology departments: a retrospective analysis

-lactamases (ESBL)-producing Escherichia coli and Enterobacter cloacae isolates from intensive care units at obstetrics & gynaecology departments: a retrospective analysis

Kun Chen¹, Guo-Liang Yang², Wen-Ping Li³, Ming-Cheng Li^4,*,†, Xue-Ying Bao^5,†

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¹ Department of Clinical Nursing, School of Nursing, Army Medical University, 400038 Chongqing, China

² Department of Ultrasound, the Second Affiliated Hospital, Army Medical University, 400037 Chongqing, China

³ Department of Obstetrics and Gynecology, DaPing Hospital, Army Medical Univerity, 400042 Chongqing, China

⁴ Department of Laboratory Diagnosis, School of Laboratory Medicine, Beihua University, 132013 Jilin, Jilin, China

⁵ Department of Laboratory Diagnosis, Associated Hospital, Beihua University, 132013 Jilin, Jilin, China

^*Correspondence: limingcheng1964@163.com; limingcheng@beihua.edu.cn (Ming-Cheng Li)
^†These authors contributed equally.

Clin. Exp. Obstet. Gynecol. 2021, 48(4), 820–827; https://doi.org/10.31083/j.ceog4804131

Submitted: 25 October 2020 | Revised: 5 January 2021 | Accepted: 22 January 2021 | Published: 15 August 2021

This is an open access article under the CC BY 4.0 license (https://creativecommons.org/licenses/by/4.0/).

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Abstract

Background: There are increasing concerns towards the transmission of extended spectrum- $\beta$ -lactamases (ESBL)-producing Enterobacteriaceae in hospital intensive care units (ICUs) at obstetrics & gynaecology departments. The aim of this study was to determine the clinical characteristics and prevalence of ESBL-producing Escherichia coli (E. coli) and Enterobacter cloacae (E. cloacae) isolates collected from ICUs at obstetrics & gynaecology departments in a tertiary care hospital, China. This study also explored the treatment options for E. coli and E. cloacae infections. Methods: E. coli and E. cloacae isolates from ICU samples were identified by using the Vitek 2 Compact System with the GN and ASTGN13 cards. Antimicrobial susceptibility profiles were determined by using the broth microdilution method. Double-disk synergy test (DDST) was performed to screen for ESBLs and combined with the EDTA-disc synergy to detect the production of carbapenemase. Enterobacterial repetitive intergenic consensus (ERIC)-PCR was applied to investigate the clonality of the isolates. Results: A total of 223 strains isolated from 283 hospitalized patients in the ICU with nosocomial infections between 2017 and 2019 were analyzed. Of these, 104 isolates were classified as E. coli and 103 isolates as E. cloacae by the VITEK GNI system. Of the 207 isolates, 131 (63.3%) were separated from sputum or tracheal secretions. ESBL-screen positive was 45.2% (47/104) for E. coli, and 44.7% (46/103) for E. cloacae. Resistance rates of ESBL-producing E. coli and E. cloacae isolates were 95.5% and 91.3% for ampicillin, respectively; 80.6% and 76.1% for ampicillin/tazobactam; 88.1% and 28.3% for ciprofloxacin; 89.6% and 15.2% for levofloxacin; 34.3% and 45.7% for netilmicin; 82.1% and 41.3% for compound sulfamethoxazole; 20.9% and 43.5% for amikacin; 58.2% and 37.0% for gentamicin; 20.9% and 69.6% for piperacillin/tazobactam. Additionally, all ESBL-producing isolates were fully resistant to cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, and aztreonam. On the other hand, isolates were fully susceptible to imipenem and meropenem. Results of ERIC-PCR in all of ESBL-producing E. coli isolates exhibited 11 distinct patterns with a similarity coefficient of 0.8. Only one distinct ERIC pattern was observed amongst the 46 strains of ESBL-producing E. cloacae. Analysis of ERIC patterns demonstrated that there was an outbreak of nosocomial infection of ESBL-producing E. coli and E. cloacae in obstetrics & gynecology ICU of this hospital. Discussion: Our data indicate that the ESBL-producing E. coli and E. cloacae are circulating in the ICU and constitute a major source infection spread. It is necessary to increase surveillance of infections in the ICU and develop adequate infection prevention strategies.

Keywords

Enterobacteriaceae

Outbreak

ESBLs

Infection control

Intensive care units

1. Introduction

Enterobacteriaceae resistance to extended-spectrum $\beta$ -lactams (ESBL) is a major public health problem worldwide [1]. Both Escherichia coli (E. coli) and Enterobacter cloacae (E. cloacae) are ESBL-producing Gram-negative Enerobacteriaceae, which are the most common cause of healthcare-associated infections (HAIs)-that comprise of hospital and community infections-admitted to intensive-care settings [2]. HAIs in intensive care units (ICUs) from ESBL-producing pathogens have become particularly problematic since they arise from the treatment and rehabilitation procedures of critically-ill patients [3]. Risk factors for infection of drug-resistant bacteria in the ICU include the following: widespread abuse, misuse, or overuse of antibiotics; the rapid renewal of antibiotics; increasing quantity and variety of pathogens present in the ICU; the diagnosis and progression of critical illnesses; undergoing surgery; the use of invasive medical devices for treatment or surgery; and prolonged stay in hospital ICUs [4, 5]. Although some research has reported a significant increase in the proportion of ESBL-positive Enterobacteriaceae including Klebsiella pneumoniae hospital infections worldwide, ICUs in China have reported only a limited number of ESBL-positive E. coli and E. cloacae isolates, especially in ICUs at obstetrics & gynaecology departments [6]. Effective and safe antimicrobial treatment is essential for treating infections in ICUs. Organizational-wide surveillance of infection-derived bacteria isolates and analysis of their susceptibility to different antimicrobial agents provides crucial information for the most effective antimicrobial therapy. Furthermore, a comprehensive analysis of epidemiology of ICU infection, and evaluating disease prognosis are essential for reducing infection mortality and morbidity in ICU [7].

This study focused on the bacterial distribution and drug resistance characteristics of ICU infections in obstetrics & gynecology departments. Furthermore, this study aimed to explore the epidemiology of pathogenic bacteria transmission and diffusion resistance by examining infectious cases at the ICU of a third-level hospital in Jilin, China between 2017 and 2019.

2. Materials and methods

2.1 Study setting and specimen collection

This study was conducted at a university-affiliated hospital, which is one of the largest hospitals in North-East China with approximately 2000 beds. This study conducted an analysis of retrospective data of Enterobacteriaceae-infected patients in three ICUs belonging to obstetrics & gynaecology settings. Two hundred and eighty-three patients who received treatment for cancer or tumor between June 2017 and June 2019 at the three ICUs were enrolled in this study. Patients with concurrent HAIs were classified according to the respiratory tract infections: pneumoniae, bloodstream infections such as catheter-related infections, urinary tract infections, surgical site infections, and other infections. For the most part, the pathogenic bacteria associated with an HAI were collected within 48 hours of hospitalization according to the local hospital criterion. Some samples were collected after 48 hours post-hospitalization. The specimens were mainly obtained from sputum or tracheal secretions, pus, blood, ascites, catheters, and drainage tubes. Multiple isolates from a single patient were excluded. The isolates from different infected sites of the same patient were also excluded. Ethical approval for collecting clinical samples was received by the institutional ethics committees of the participating hospital. Informed consent forms were reviewed and signed by all participants before sample collection.

2.2 Identification and detection of resistance to 17 antibiotics agents

E. coli and E. cloacae isolates were identified by using the Vitek 2 Compact System with GN and ASTGN13 cards (bioMérieux, Marcy l’Etoile, France). Susceptibility to a panel of 17 antimicrobial agents was assessed by using the broth microdilution method according to the recommendations by the Clinical and Laboratory Standards Institute (CLSI, 2012) [8]. The following antibiotics (AB Biodisk, Solna, Sweden) were tested: ampicillin, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, levofloxacin, netilmicin, aztreonam, ciprofloxacin, amikacin, gentamicin, imipenem, meropenem, ampicillin/tazobactam, piperacillin/tazobactam and compound sulfamethoxazole.

E. coli ATTCC 25922 was the susceptible control strain, and K. pneumoniae 700603 and E. cloacae ATCC 13047 were the ESBL-positive control strains. K. pneumoniae A1500 was the carbapenemase-positive control strains.

2.3 Determination of ESBL-producing and carbapenemase-producing strains

Double-disk synergy test (DDST) was performed to screen for ESBLs and combined with the EDTA-disc synergy to detect carbapenemase. Suspected ESBL-producing strains were screened using cefotaxime (Oxoid, Basingstoke, UK) (30 $\mu$ g) and ceftazidime (Oxoid) (30 $\mu{}$ g) in combination with clavulanic acid (Oxoid) (10 $\mu{}$ g) according to the CLSI recommended disk diffusion method [8]. The screening test was considered positive if the inhibitory zone diameter to cefotaxime/clavulanic acid or ceftazidime/clavulanic acid exhibited an increase of more than 5 mm. For EDTA-disc synergy to detect production of carbapenemase, a 10 $\mu{}$ g imipenem disc (AB Biodisk, Solna, Sweden) and a blank filter disc containing 10 $\mu{}$ L of 0.5 M EDTA solution (Oxoid) were placed 10 mm between the disc edges on a Mueller-Hinton agar plate whose surface was inoculated with the test strains. The presence of an enlarged inhibition zone was considered to be positive test result.

2.4 Clonality analysis of E. coli and E. cloacae isolates by ERIC

2.4.1 Extraction of total DNA from E. coli and E. cloacae isolates

A single colony was selected from the passage medium and incubated overnight at 37 ${}^{\circ}{}$ C after being added to a test tube containing 2 mL of Luria-Bertani liquid. The next day, 2 mL of bacterial liquid was centrifuged at 12,000 r/min for five minutes. The supernatant was discarded and added to 400 $\mu{}$ L ddH ${}_{2}$ O and boiled for 10 minutes after mixing. The mixture was then cooled and centrifuged at 12,000 r/min for five minutes, and the supernatant was absorbed and stored at -20 ${}^{\circ}{}$ C.

2.4.2 ERIC-PCR amplification

The primer sequences were P1-ATGTAAGCTCCTGGGGATTCAC and P2-AAGTAAGTGACTGGGGTGAGCG. The system contained 2 $\mu{}$ L of P1 and P2 primers, and the DNA template solution at 3 $\mu{}$ g/L. ddH ${}_{2}$ O was added to the total reaction system of 50 $\mu{}$ L. PCRs were conducted in a GeneAmp PCR system 9600 (Perkin-Elmer, Waltham, MA, USA) under the following reaction conditions: denaturation at 94 ${}^{\circ}$ C for five min, denaturation at 94 ${}^{\circ}$ C for 30 s, annealing at 56~58 ${}^{\circ}$ C for 45 s, extension at 72 ${}^{\circ}$ C for 30 s, and 32 cycles at 72 ${}^{\circ}$ C after five min. PCR products were analyzed by 2% agarose gel electrophoresis at 60 V for 40 min. A molecular weight DNA marker from 100~600 bp was used as a reference and a gel imaging analysis system was used to observe and analyze the results. Band comparisons were carried out by clustering analysis with the unweighted pair group method using Quantity One (Version 4.6.2) (Bio-Rad Laboratories, Hercules, CA, USA). Isolates were considered as the same origins if their similarity coefficients were equal to or more over 0.8, whereas similarity coefficients below 0.8 meant that isolate origins were dissimilar [9].

2.5 Statistical analysis

Differences in drug resistance rates of non-ESBL-producing and ESBL-producing strains were tested by Chi-square test. All drug resistant data were analyzed using SPSS version 13.0 (IBM Corp., Armonk, NY, USA). Analyses with a value of P $<$ 0.05 were considered to be statistically significant.

3. Results

3.1 Epidemiological characteristics of specimens and isolates

From June 2017 to June 2019, a total of 283 patients from three ICUs were enrolled in this study to estimate the quantity and types of infections present in this population. These patients received treatment or surgery for cancer or tumor at a median age of 63 years. Of the 283 samples, 104 were classified as E. coli and 103 as E. cloacae by the VITEK GNI system. The remaining samples were classified as other Enterobacteriaceae and non-Enterobacteriaceae, which were not included in this study (data not shown).

The clinical distribution of the specimens was mainly composed of sputum or tracheal secretions (n = 179, 63.3%), followed by skin and purulent infections (n = 33, 11.5%), blood (n = 43, 15.2%), ascites (n = 20, 7.1%), and catheters and drainage tubes (n = 8, 2.8%).

3.2 Antimicrobial susceptibility analysis

Resistance frequencies of ESBL-producing E. coli and E. cloacae isolates based on CLSI microdilution demonstrated that 67 (64.4%) strains of E. coli and 46 (44.7%) strains of E. cloacae isolates were ESBL-positive. No isolates were resistant to carbapenem.

Resistance rates of ESBL-producing E. coli and E. cloacae isolates were 95.5% and 91.3% for ampicillin, respectively; 80.6% and 76.1% for ampicillin/tazobactam; 88.1% and 28.3% for ciprofloxacin; 89.6% and 15.2% for levofloxacin; 34.3% and 45.7% for netilmicin; 82.1% and 41.3% for compound sulfamethoxazole; 20.9% and 43.5% for amikacin; 58.2% and 37.0% for gentamicin; and 20.9% and 69.6% for piperacillin/tazobactam. All of ESBL-producing isolates were 100% resistant to cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, and aztreonam. The susceptibilities of isolates to imipenem and meropenem were 100%.

The susceptibilities of non-ESBL-positive E. coli and E. cloacae isolates were 89.2% and 91.3% to ampicillin, respectively; 97.3% and 96.5% to ampicillin/tazobactam; 94.6% and 94.7% to cefazolin; 89.2% and 96.5% to cefuroxime; 97.3% and 98.3% to ceftazidime; 97.3% and 98.3% to ceftriaxone; 91.9% and 92.3% to cefepime; 89.2% and 100.0% to aztreonam; 32.4% and 68.4% to ciprofloxacin; 37.8% and 91.2% to levofloxacin; 70.3% and 56.1% to netilmicin; 43.2% and 61.4% to compound sulfamethoxazole; 62.2% and 71.9% to amikacin; 43.2% and 68.4% to gentamicin; and 97.3% and 93.0% to piperacillin/tazobactam. The susceptibilities to imipenem and meropenem were the only ones at 100%. The resistance rates to different antibiotics between ESBL-positive and non-ESBL-positive isolates are summarized in Tables 1 and 2.

Table 1.Susceptibility to common antimicrobials of 104 E. coli isolates.

Antimicrobiol agents	ESBL positive (n = 67)		ESBL negative (n = 37)		X ${}^{2}$	P value
Antimicrobiol agents	R (%)*	S (%)	R (%)	S (%)	X ${}^{2}$	P value
Ampicillin	64 (95.5)	3 (4.5)	4 (10.8)	33 (89.2)	4.42	$<$ 0.005
Ampicillin/azobactam	54 (80.6)	13 (19.4)	1 (2.7)	36 (97.3)	5.01	$<$ 0.005
Cefazolin	67 (100.0)	0 (0.0)	2 (5.4)	35 (94.6)	130.67	$<$ 0.005
Cefuroxime	67 (100.0)	0 (0.0)	4 (10.8)	33 (89.2)	169.60	$<$ 0.005
Ceftazidime	67 (100.0)	0 (0.0)	1 (2.7)	36 (97.3)	24.34	$<$ 0.005
Ceftriaxone	67 (100.0)	0 (0.0)	3 (8.1)	34 (91.9)	83.79	$<$ 0.005
Cefepime	67 (100.0)	0 (0.0)	3 (8.1)	34 (91.9)	19.51	$<$ 0.005
Aztreonam	67 (100.0)	0 (0.0)	4 (10.8)	33 (89.2)	88.48	$<$ 0.005
Ciprofloxacin	59 (88.1)	8 (11.9)	25 (67.6)	12 (32.4)	3.31	$>$ 0.005
Gentamicin	39 (58.2)	28 (41.8)	21 (56.8)	16 (43.2)	3.80	$<$ 0.005
Imipenem	0 (0.0)	67 (100.0)	0 (0.0)	37 (100.0)	2.01	$>$ 0.005
Meropenem	0 (0.0)	67 (100.0)	0 (0.0)	37 (100.0)	0	0
Levofloxacin	60 (89.6)	7 (10.5)	23 (62.2)	14 (37.8)	4.69	$<$ 0.005
Netilmicin	23 (34.3)	44 (65.7)	11 (29.7)	26 (70.3)	4.91	$<$ 0.005
Compound sulfamethoxazole	55 (82.1)	12 (17.9)	21 (56.8)	16 (43.2)	61.68	$<$ 0.005
Piperacillin/tazobactam	14 (20.9)	53 (79.1)	1 (2.7)	36 (97.3)	4.54	$<$ 0.005
Amikacin	20 (29.9)	47 (70.2)	14 (37.8)	23 (62.2)	4.74	$<$ 0.005
Note: *P $<$ 0.05 compared with the ESBL negative group.

Table 2.Susceptibility to common antimicrobials of 103 E. cloacae isolates.

Antimicrobiol agents	ESBL positive (n = 46)		ESBL negative (n = 57)		X ${}^{2}$	P value
Antimicrobiol agents	R (%)*	S (%)	R (%)	S (%)	X ${}^{2}$	P value
Ampicillin	42 (91.3)	4 (8.7)	4 (7.0)	53 (93.0)	4.42	$<$ 0.005
Ampicillin/azobactam	35 (76.1)	11 (23.9)	2 (3.5)	55 (96.5)	4.52	$<$ 0.005
Cefazolin	46 (100.0)	0 (0.0)	3 (5.3)	54 (94.7)	130.67	$<$ 0.005
Cefuroxime	46 (100.0)	0 (0.0)	2 (3.5)	55 (96.5)	169.60	$<$ 0.005
Ceftazidime	46 (100.0)	0 (0.0)	1 (1.8)	56 (98.3)	24.34	$<$ 0.005
Ceftriaxone	46 (100.0)	0 (0.0)	4 (7.0)	53 (93.0)	83.79	$<$ 0.005
Cefepime	46 (100.0)	0 (0.0)	2 (3.5)	55 (96.5)	19.51	$<$ 0.005
Aztreonam	46 (100.0)	0 (0.0)	0 (0.0)	57 (100.0)	88.48	$<$ 0.005
Ciprofloxacin	13 (28.3)	33 (71.7)	18 (31.6)	39 (68.4)	3.31	$>$ 0.005
Gentamicin	17 (37)	29 (63.0	20 (35.1)	37 (64.9)	3.80	$<$ 0.005
Imipenem	0 (0.0)	46 (100.0)	0 (0.0)	57 (100.0)	2.01	$>$ 0.005
Meropenem	0 (0.0)	46 (100.0)	0 (0.0)	57 (100.0)	0	0
Levofloxacin	7 (15.2)	39 (84.8)	5 (8.8)	52 (91.2)	4.69	$<$ 0.005
Netilmicin	21 (45.7)	25 (54.4)	25 (43.9)	32 (56.1)	4.91	$<$ 0.005
Compound sulfamethoxazole	19 (41.3)	27 (50.7)	22 (38.6)	35 (61.4)	61.68	$<$ 0.005
Piperacillin/tazobactam	32 (69.6)	14 (30.4)	4 (7.0)	53 (93.0)	4.21	$<$ 0.005
Amikacin	20 (43.5)	26 (56.5)	16 (28.1)	41 (71.9)	4.27	$<$ 0.005
Note: *P $<$ 0.05 compared with the ESBL negative group.

Next, this study investigated a local difference of antibiotic resistance between ESBLs and non-EBSL strains. This study found a significant difference between non-ESBL-producing strains and ESBL-producing strains (P $<$ 0.05) for the exception of susceptibility to ciprofloxacin (P $>$ 0.05).

3.3 Isolate clonality analysis by ERIC-PCR

Isolate clonality analysis of all ESBL-producing strains was conducted by ERIC-PCR typing. One distinct ERIC profile was observed amongst 46 strains of ESBL-producing E. cloacae, showing that these isolates were similar to clones (Fig. 1). A representative of the same band was selected in 67 ESBL-producing E. coli isolates from different samples for dendrogram cluster analysis. This analysis revealed 11 distinct patterns with a similarity coefficient of 0.8, indicating that these isolates have similar origins. A clonal association was found between these strains: 54 (80.6%) originated from identical clones (Figs. 2 and 3), indicating that there is an outbreak at the study setting. Furthermore, the medical history of ICU patients with the same drug-resistant strains was similar. The majority of drug-resistant strains with identical clones infected the lower respiratory tract. Approximately 76% of ICU patients had a history of mechanical ventilation.

Fig. 1.

Representative gel showing banding profiles by ERIC-PCR analysis in ESBLs-producing E. cloacae isolates.
Note:
M: DNA molecular weight; 1~13: ESBLs-producing E. cloacae isolates from different samples in ICU. 1-2: strains isolated from pus; 3-4: strains isolated from urine; 5-6: strains isolated from blood; 7: strains isolated from ascite; 8-9: strains isolated from cathers and drainage tube; 11-13: strains isolated from sputum or tracheal secretions. ERIC, Enterobacterial repetitive intergenic consensus.

Fig. 2.

Representative gel showing banding profiles by ERIC-PCR analysis in ESBLs-producing E. coli isolates.
Note:
M: DNA molecular weight; 1~13: ESBLs-producing E. coli isolates from different samples in ICU. 1-2: strains isolated from pus; 3-4: strains isolated from urine; 5-6: strains isolated from blood; 7: strains isolated from ascite; 8-9: strains isolated from cathers and drainage tube; 11-13: strains isolated from sputum or tracheal secretions; ERIC, Enterobacterial repetitive intergenic consensus.

Fig. 3.

Dendrogram from ERIC-PCR analysis in ESBLs-producing E. coli isolates.
Note:
The scale bar showed the similarity values. Isolates were considered as the same origins if their similarity coefficients were equal to or more over 0.8, whereas, lower 0.8 is different origins. ERIC, Enterobacterial repetitive intergenic consensus.

4. Discussion

Antibacterial drugs have been administered extensively in health care for the treatment of disease, especially for critically ill patients in the ICU. However, the incidence of infections resistant to ESBL-producing Enterobacteriaceae has rapidly increased in recent years [10]. Research has documented many instances of outbreaks of HAIs in ICUs caused by ESBL-producing E. coli and E. cloacae[11].

This study collected samples from ICU patients and found that the prevalence of E. coli and E. cloacae isolates among ESBL-producing Enterobacteriaceaewas estimated at 64.4% and 44.7%, respectively. These isolates were most common in respiratory tract and skin infections. The results of this study indicated that female patients with a tumor or cancer were likely to be infected by ESBL-producing Enterobacteriaceae due to their immunological status or the excessive use of $\beta$ -lactams. This finding agrees with other research that isolates from ICUs in China contain E. coli and Klebsiella pneumoniae in addition to E. cloacae [12, 13]. E. coli and E. cloacae belong to a group of conditional pathogens that are part of the normal intestinal flora, but can cause infections of the respiratory and urinary tracts [14, 15]. An epidemiological study reported high rates of E. coli and E. cloacae producing ESBLs in Singapore (44%) and China (37%) [16]. Another research indicates that both ESBL-producing E. coli and E. cloacae are closely related to antibacterial drug resistance. The same bacterium is likely to carry a variety of ESBLs, which may cause multi-drug resistance [17, 18].

ESBL-producing strains of E. coli and E. cloacae isolates showed statistically higher resistance rates to cephalosporins than non-ESBL-producing strains. In contrast, there was no obvious difference in the resistance to ciprofloxacin between ESBL-producing and non-ESBL-producing strains. Comparing the sensitivity of cephalosporin antibiotics with sulbactam and tazobactam, drug resistance in ESBL-producing strains was much lower than cephalosporins alone. The findings of this study demonstrate that antibiotic resistance in E. coli and E. cloacae can be reduced if ESBL production is addressed. All ESBL-producing strains exhibited high resistance to cephalosporins, while at the same time showed high sensitivity to carbapenems. Because the northeast part of China is the coldest region in the country with the highest incidence of respiratory system diseases, overuse of antimicrobial drugs with the exception of carbapenems might explain why this study found a difference in antimicrobial agent susceptibility. Additionally, the findings showed that ESBL-producing bacteria were more resistant to aminoglycosides and levofloxacin than those with high resistance to cephalosporin antibiotics. Resistance to synthetic antibiotics such as sulfonamides was also lower than resistance to ampicillin.

The initial use of advanced cephalosporin antibiotics, especially the unreasonable use of third generation cephalosporins, marks the beginning of habitual therapy. Habitual therapy may cause health care providers to ignore the role of aminoglycosides and other antibacterial drugs that may contribute to resistance to drugs. ESBL-producing E. coli and E. cloacae isolates transiently colonize on the hands of hospital staff, increasing the likelihood of horizontal transmission between patients. ESBL-producing Enterobacteriaceae were frequently accompanied by resistance to an array of antibiotics. Studies have shown that multiple drug resistance mechanisms exist for E. coli and E. cloacae including mutations in Amp C enzymes and porin loss [19, 20]. However, the molecular mechanism of resistance was outside the scope of this study and future research may consider paying attention to this research priority.

Clinically, the common utilization of high-grade cephalosporin antibiotics for the treatment of ICU patients with nosocomial infections alongside the unreasonable use of third-generation cephalosporins may have caused the large number of drug-resistant strains that were found in the current study [21, 22]. As a result, patients who are ineffectively treated with advanced cephalosporins or $\beta$ -lactamase inhibitors due to antimicrobial resistance may only be treated by carbapenems [23].

In the present investigation, both E. coli and E. cloacae were highly sensitive to carbapenems. No significant difference in resistance was found between ESBL-producing and non-ESBL-producing strains, indicating that carbapenems could be used to treat ESBL-resistant strains. Carbapenems, which are atypical $\beta$ -lactam antimicrobial agents, have the strongest antimicrobial activity and the broadest antimicrobial spectrum. The low toxicity and high stability of $\beta$ -lactamase makes them an important target for the treatment of severe infections, especially when other antimicrobial agents are ineffective [24, 25]. High sensitivity of ESBL-producing E. coli isolates and ESBL-producing E. cloacae isolates to carbapenems might be explained by the fact that carbapenem has been seldom administered in China compared to other drugs. However, in contrast to data collected from health service organizations in East China, reduced susceptibilities of E. coli and E. cloacae indicate a local resistance to carbapenem [26]. Taken together, carbapenem overuse might explain why there might be a difference between areas with patients from a higher socioeconomic status than those areas with lower socioeconomic status [27].

In the epidemiological analysis of nosocomial infections in the ICU, ERIC-PCR was used to identify isolates based on ERIC-PCR fingerprints. Since the introduction of the ERIC-PCR technique by Versalovic et al. in 1991, the technique has been critical for the epidemiological investigation of Gram-negative bacteria [28]. Results of ERIC-PCR performed in this study found 11 distinct profiles across ESBL-producing E. coli isolates, and one distinct profile across 46 strains of ESBL-producing E. cloacae. Overall, ERIC profiles demonstrated that there is an outbreak of nosocomial infection (ESBL-producing E. coli and E. cloacae) in the ICUs of the hospital.

Interestingly, this study found that all 46 ESBL-producing E. cloacae strains belonged to the same clonal type. However, there was variation in the antimicrobial profile for netilmicin, sulphamethaxazole, piperacillin/tazobactam, and amikacin. These four antibiotics were seldom used in the study hospital. It is possible that the encoding resistant genes for these antibiotics were primarily carried by mobile genetic elements, such as transposons and introns on a plasmid. Evidence from this study illustrated that plasmid mediating ESBLs might produce other resistant gene expressions, which may be associated with an increased use of antibiotics. Moreover, the E. cloacae complex encompasses several species comprising of 12 genetic clusters [29]. This study found significant variation in the four antimicrobial profiles among these isolates from ICU across different years of this study. This may be due to shifting or point mutations in these mobile genetic structures that may exert a pressure for selection [30, 31].

In order to control the production and spread of ESBLs, health care providers may consider careful and reasonable use of antibacterial drugs. For example, health care providers might consider limiting the empirical routine application of high-level antibiotics. Education and awareness surrounding the appropriate use of antibacterial drugs amongst health care workers and patients should be strengthened alongside improving and clarifying antibiotic use guidelines in pharmacies and hospital ICUs [32]. These measures could effectively mitigate or reduce the spread of infection of ESBLs bacteria. Overall, this study emphasizes the need for medical staff to use antimicrobial agents rationally [33].

This study has some limitations. First, genotypic or molecular data of all strains were not documented. Second, the molecular epidemiology of isolates was not included. Future research may consider focusing on genetic types and the mechanism of transmission.

5. Conclusions

The findings of this study indicate that the ESBL-producing E. coli and E. cloacae clones are circulating in the ICU at obstetrics & gynaecology departments and constitute a major source of infection at a large hospital in China. This study also found that carbapenems may be a reasonable choice in the treatment of ESBL-producing bacteria. In summary, there is a strong need for increased hospital-wide surveillance and the development of adequate infection prevention strategies.

Abbreviations

CLSI, the Clinical and Laboratory Standards Institute; DDST, Double-disk synergy test; E. cloacae, Enterobacter cloacae; E. coli, Escherichia coli; ERIC, Enterobacterial repetitive intergenic consensus; ESBL, extended-spectrum $\beta$ -lactams; HAIs, healthcare-associated infections; ICU, intensive care unit.

Author contributions

KC conceived, designed the experiments and wrote a draft manuscript. MCL and XYB analyzed, interpreted the results of the experiments and revised the manuscript. GLY and WPL performed the experiments. XYB collected the clinical data. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Ethical approval for collecting clinical samples was received by the institutional ethics committees of the participating hospital. Informed consent forms were reviewed and signed by all participants before samples collection (Ethical approval number: Protocol Number 2019-01-01).

Acknowledgment

We thank three anonymous reviewers for excellent criticism of the article.

Funding

The present project was a part of National Natural Science Foundation of China project (81402979), the Jilin Science and Technology Development Program (20140307008YY and 2014C33155); the Health and Family Planning Commission of Jilin Province (2018J098) also supported the project.

Conflict of interest

The authors declare no conflict of interest.

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