Antimicrobial resistance and epidemiology of ESBLs-producing Escherichia coli and Enterobacter cloacae isolates from the intensive care unit in an a liated hospital of University, China


 Background：Concerns are increasing over the importance of the hospital intensive care units (ICU) for the transmission of extended spectrum-β-lactamase (ESBLs) -producing Enterobacteriaceae. We reported the clinical characteristics and epidemiology of ESBLs isolates collected from a tertiary care hospital in China. Methods：Escherichia coli（E. coli）and Enterobacter cloacae （E. cloacae）isolates from ICU infection samples were isolated and identified. Antimicrobial susceptibility profiles and production of ESBLs were determined by using the disk diffusion method and the broth microdilution method. Clonality of isolates was determined by ERIC-PCR techniques. Results：From the included the 223 strains isolated from hospitalized patients with nosocomial infections in ICU during 2016 to 2018, the majority of isolates belonged to Gram-negative Enerobacteriaceae including E. coli (46.6% of all strains), and E. cloacae (46.2% of all strains). 63.25% of samples were separated from sputum or tracheal secretions. All of 207 isolates, ESBL-screen positive E. coli was 45.2% (47/104), and 44.7% (46/103) for E. cloacae. Resistance rates of ESBLs-producing E. coli and E. cloacae isolates were 95.5%-91.3% for ampicillin, 80.6%-76.1% for ampicillin/azobactam, 88.1%-28.3% for ciprofloxacin, 89.6%-15.2% for levofloxacin, 34.3%-45.7% for netilmicin, 82.1%-41.3% for compound sulfamethoxazole, 20.9%-43.5% for amikacin, 58.2%-37.0% for gentamicin, 20.9%-69.6% for piperacillin/tazobactam. All of ESBLs-producer isolates resistant to cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime in additon to aztreonam were 100%, whereas the susceptibilities of isolates to imipenem and meropenem were 100%. Results of ERIC-PCR in all of ESBLs-producing E. coli isolates exhibited 11 distinct patterns using a similarity coefficient of 0.8. And one distinct ERIC profiles were observed amongst 46 strains of ESBLs-producing E. cloacae. ERIC profiles demonstrated an outbreak of nosocomial infection and ESBLs-producing E. coli and E. cloacae prevalent in the ICU of this hospital.Conclusions：Our data indicate that the ESBLs-producing E. coli and E. cloacae clones are circulating in the ICU and constitute a major source for further disseminating in this hospital. It is necessary to increase surveillance and development of adequate prevention strategies.


Introduction
The incidence of infections due to the emergence and dissemination of Enerobacteriaceae has rapidly increased for the last several decades. Resistance of Enerobacteriaceae to extended-spectrum β-lactams worldwide is becoming a major public health problem [1]. Both Escherichia coli (E. coli) and Enterobacter cloacae (E. cloacae) presently are the ESBL producing Gram-negative Enerobacteriaceae, which have emerged as the most common cause of hospital-and community-acquired infection admitted to neonatal intensive-care settings [2]. The nosocomial infections of the intensive care units (ICU) that occur in the speci c environment in which the treatment and rehabilitation of critically ill patients is frequently Identi cation and detection of resistance to 17 antibiotics agents The identi cation of E. coli and E. cloacae was identi ed by using the Vitek 2 Compact System with GN card and ASTGN13 card (bioMérieux, Marcy l'Etoile, France). Susceptibility to a panel of 16 antimicrobial agents was determined according to Clinical and Laboratory Standards Institute recommendations (CLSI, 2012) [8]. The tested antibiotics (AB Biodisk, Solna, Sweden) were included: ampicillin, cefazolin, cefuroxime, ceftazidime, ceftriaxone, cefepime, levo oxacin, netilmicin, aztreonam, cipro oxacin, amikacin, gentamicin, imipenem, meropenem, ampicillin /azobactam, piperacillin / tazobactam and compound sulfamethoxazole.
MIC values for 17 drugs at 12 concentrations in vitro were determined using a micro broth dilution method. E. coli ATTCC 25922, K. pneumoniae 700603 (ESBL positive) and E. cloacae ATCC 13047 were used as quality control strains.
Clonality analysis of E. coli and E. cloacae isolates by ERIC Total DNA extraction from E. coli and E. cloacae A single colony was selected from the passage medium and incubated overnight at 37 ° 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 5 minutes. The supernatant was discarded and added to 400 µl dd H 2 O and boiled for 10 minutes after mixing. The mixture was cooled and centrifuged at 12, 000 r/min for 5 minutes, and the supernatant was absorbed and stored at − 20° C.

ERIC-PCR ampli cation
The primer sequences were P1-ATGTAAGCTCCTGGGGATTCAC and P2-AAGTAAGTGACTGGGGTGAGCG. The system contained 2 µl of P1 and P2 primers, and the DNA template solution at 3 µg/l. ddH 2 O was added to the total reaction system of 50 µl. PCRs were conducted in a GeneAmp PCR system 9600 (Perkin-Elmer, USA) under the following reaction conditions; denaturation at 94 ℃ for 5 min, denaturation at 94 ℃ for 30 s, annealing at 56 58 ℃ for 45 s, extension at 72 ℃ for 30 s, and 32 cycles at 72 ℃ after 5 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 for 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). Isolates were considered as the same origins if their similarity coe cients were equal to or more over 0.8, whereas, lower 0.8 is different origins according to the reference [9].

Statistical analysis
Differences in the drug resistance rates of non-ESBLs-producing and ESBLs-producing strains were tested by Chi-square test. All drug resistant data were analyzed using SPSS13.0 statistical software. A value of P < 0.05 was considered as statistically signi cant. The clinical distribution of the specimens was mainly composed of sputum or tracheal secretions accounting for 63.3% of the samples, followed by skin and purulent infections(11.5%), blood 15. 2% , ascites 7.1% , catheters and drainage tubes 2.4% and cerebrospinal uid 0.5% .

Determination of ESBLs phenotypes
In the primary screening test, 72 strains of E. coli in the inhibitory zone to ceftazidime with a diameter of ≤ 22 mm, and the inhibitory zone to ceftriaxone, which was ≤ 25 mm in diameter, were highly suspected ESBLs-producing bacteria. It was con rmed that 67 strains of E. coli alone were detected in the inhibitory zone to cephalosporin which had a diameter of ≥ 5 mm. At the same time, the diameter of the inhibitory zone to clavulanic acid was ≥ 5 mm.
All of 103 strains of E. cloacae, 61 strains of E. cloacae to ceftazidime and ceftriaxone were highly suspected to produce ESBLs in the screening test. The inhibitory zone to ceftazidime with a diameter of ≤ 22 mm and the inhibitory zone to ceftriaxone which was ≤ 25 mm in diameter were highly suspected to produce ESBLs. It was con rmed that there were 46 strains of E. cloacae isolates whose inhibitory zone diameter to cephalosporin was ≥ 5 mm. Meanwhile, the diameter of the inhibitory zone to clavulanic acid was ≥ 5 mm.

Clonality of isolates analysis by ERIC-PCR
Clonality of isolates analysis of all the ESBLs-producing strains was investigated by ERIC-PCR typing. Based on the ERIC-PCR typing, one distinct ERIC pro les were observed amongst 46 strains of ESBLsproducing E. cloacae, showing that these isolates were of the equal clones (Fig. 1). We selected a representative of the same band in 67 ESBLs-producing E. coli isolates from different samples for dendrogram cluster analysis, revealed 11 distinct patterns using a similarity coe cient of 0.8. An obvious clonal association was found within these strains, of which 54 (80.6%) were of the identical clones ( Figs. 2 and 3), indicating an outbreak situation. The history of the same drug-resistant strains in these ICU patients was similar. Most of the drug-resistant strains with the same clones had more infections in the lower respiratory tract, and 76% of patients had a history of mechanical ventilation.

Discussion
Antibacterial drugs have been administered extensively in clinic, especially in the treatment of ICU patients. However, the widespread use of antibacterial drugs produces further resistant bacteria, which is becoming a serious public health concern [10]. The abuse of this antibacterial drug causes the body to produce multi-drug resistance and the risk of super bacterial infection increases. The rational application of antibacterial drugs in the clinic effectively prevents the spread of drug-resistant strains and more comprehensive understanding of drug resistance mechanisms, and epidemiology of drug-resistant strains has much potential to improve the cure rates of ICU patients [11].
The majority of ICU isolates collected in the participating multicenters consisted of E. coli and Klebsiella pneumoniae in additon to E. cloacae in China [12,13]. In this study, ESBLs-producing and non-ESBLsproducing strains of E. coli showed signi cant differences in resistance to cephalosporins with ESBLsproducing strains having signi cantly higher resistance rates. In contrast, there was no obvious difference between the ESBLs-producing and non-ESBLs-producing strains concerning cipro oxacin. In the comparison to the sensitivity of cephalosporin antibiotics with sulbactam and tazobactam, the drug resistance was extremely lower than that of cephalosporins alone. This group of studies fully demonstrated that if the production of ESBLs can be reduced as much as possible, the resistance of E. coli can also be greatly reduced.
E. coli and E. cloacae belong to conditional pathogens that are part of the normal intestinal ora but can cause infections of the respiratory and urinary tracts [14,15]. An epidemiological study has reported that the detection rate of E. coli and E. cloacae producing ESBLs was increasing, 44% in Singapore, 37% in China [16]. Both E. coli and E. cloacae producing ESBLs are closely related to the drug resistance of antibiotics, and the same type of bacteria are likely to carry a variety of ESBLs, leading to multi-drug resistance [17,18]. Studies have also shown that there are multiple drug resistance mechanisms in E. coli and E. cloacae, which include mutations in Amp C enzymes and porin loss, which has been described in previous literatures [19,20]. However, we did not explore molucular mechanisms of resistances.
In this study, we collected samples from different parts of the patients and found that E. coli and E. cloacae were most common in infections of the respiratory tract and skin. The resistance of E. cloacae to 17 antimicrobial agents was similar to that of E. coli. All of them exhibited high resistance to cephalosporins, whilst they were highly sensitive to carbapenems. Because the northeast part of China is the most colded region with the highest incurrence of respiratory system diseases, antimicrobial overuse might be an explanation for the antimicrobial agents susceptibility difference in this area.
Clinically, the application of a large number of high-grade cephalosporin antibiotics in the treatment of ICU patients with nosocomial infections and unreasonable use of the third-generation cephalosporins have resulted in a large number of drug-resistant strains [21,22]. Patients who are ineffectively treated with advanced cephalosporins or β-lactamase inhibitors can only further use carbapenem antibacterial drugs in treating Gram-negative bacteria [23].
Carbapenems have the strongest antimicrobial activity, broadest antimicrobial spectrum and belong to atypical beta-lactam antimicrobial agents. The low toxicity and high stability of beta-lactamase make them one of the best options for the treatment of severe infections when other antimicrobial agents are ineffective [24,25].
In this study, both E. coli and E. cloacae were highly sensitive to carbapenems, and no signi cant difference in resistance between ESBLs-producing and non-ESBLs-producing strains was observed, indicating that carbapenems could be used as an option in the treatment of ESBLs-resistant strains. In contrast to data collected from centers in east China, the reduced susceptibilities of E. coli and E. cloacae indicated a local carbapenem resistance [26]. Taken together, the carbapenem overuse might be an explanantion for the difference because of the developed areas with the higher incomes [27].
In the epidemiological analysis of nosocomial infections in the ICU, ERIC-PCR was used to identify  [28,29]. ERIC pro les demonstrated an outbreak of nosocomial infection and ESBLsproducing E. coli and E. cloacae prevalent in the ICU of this hospital.
The study found that ESBLs-producing bacteria were resistant to aminoglycoside antibiotics such as levo oxacin, and were more resistant to the above two types of antibacterial drugs than those with high resistance to cephalosporin antibiotics. The resistance to synthetic antibiotics such as sulfonamides was also lower than that of simple ampicillin. In the clinical application of antibiotics, the initial use of advanced cephalosporin antibiotics, especially the unreasonable use of the third generation cephalosporins, is the beginning of habitual therapy. These practices cause us to ignore the role of aminoglycosides and other antibacterial drugs that lead to common clinical drug resistance. In order to control the production and spread of ESBLs from the source, more reasonable use of antibacterial drugs should be followed by limiting the routine empirical application of high-level antibiotics by medical personnel. Publicity and education on the use of antibacterial drugs amongst health care workers should be strengthened along with improvements in the management of pharmacies and hospital ICUs [30,31]. These measures could effectively cut off the source of infection and a mode of transmission of ESBLs bacteria. In addition, this could guide medical staff to rationally use antimicrobial agents [32]. This study has some limitations. Firstly, the genotypic or molecular data of all strains were not documented. Secondly, the molecular epidemiology was not included. Further studies are needed to con rm the genetic types and the mechanism of transmission.

Conclusions
our ndings indicate that the ESBLs-producing E. coli and E. cloacae clones are circulating in the ICU and constitute a major source for further disseminating in this hospital. Carbapenems are the reasonable choice in the treatment of ESBLs-producing bacteria. It is necessary to increase surveillance and development of adequate prevention strategies. Coverletter.doc