The prevalence of Escherichia coli strains with extended spectrum beta-lactamases isolated in China

The extended-spectrum-lactamases-producing Escherichia coli has rapidly spread worldwide. Escherichia coli has been becoming much more resistant to β-lactam antibiotics and other commonly available antimicrobials. We investigated the prevalence, resistance, and probable gene type of extended spectrum beta-lactamases (ESBLs) using minimum inhibitory concentrations (MICs) testing and polymerase chain reaction (PCR). We have collected 289 single-patient E. coli Isolates based on samples of China from July 2013 to August 2014. This article explored that the prevalence of ESBL-producing Isolates showed multi-resistant to antimicrobials such as fluoroquinolones, trimethoprim, tetracycline and aminoglycosides, and so on. The frequencies of resistance in Isolates were as follows: Ciprofloxacin, 74%, gentamicin, 69.5%, levofloxacin, 63%, tobramycin, 39%, and minocycline, 7.9%. According to our results, 197(68.2%) of the total 289 Isolates were ESBL-producing strains; further, 172 (87.3%) producers contained genes encoding CTX-M enzymes and 142(72.1%) producers contained genes encoding TEM enzymes. Most ESBL-producing Escherichia coli has produced more than one type of β-lactamase. Nucleotide sequence analysis has revealed the diversity of ESBLs types: CTX-M -15 is in the majority and TEM-135, CTX-M-3, CTX-M-98, CTX-M-14, CTX-M-142, CTX-M-65, CTX-M-55, CTX-M-27, and CTX-M-123 have been recovered. The results confirm that ESBL producers which are common in hospital strains of Escherichia coli are resistant to cephalosporins and other antibiotics in China. It is important to monitor such strains closely and provide scientific evidence of rational application of antibiotics to prevent their spread.


Introduction
The resistance of ESBL-producing Enterobacteriaceae has currently become one of the most important nosocomial resistance problems around the world and it has been found that there are much more community-acquired ones (Bradford, 2001;Valverde et al., 2004). Escherichia coli has been a universal commensal bacterium causing infections in humans and animals and often leads to urinary tract infections (UTI) and bacteremia for humans (Paterson and Bonomo, 2005). ESBL-producing Enterobacteriaceae were firstly reported in the middle of 1980s in Germany, since then a steady increase of these strains have been reported worldwide (Bradford, 2001).
Recently, researchers have paid much more attention to extended spectrum beta-lactamases (ESBLs). As well known, the production of β-lactamase is the most common mechanism of bacterial resistance to β-lactam antibiotics; with the introduction of extended-spectrum cephalosporins, ESBLs has spread across the world (Medeiros, 1997;Yagi et al., 2000;Saladin et al., 2002;Yong and Toleman, 2009). ESBLs could inactivate oxyimino-βlactams like third-generation cephalosporins and aztreonam, but not hydrolyze the carbapenems, which are highly susceptible to inhibition by clavulanic acid and tazobactam (Livermore, 1995). ESBL-producing strains are not only highly resistant to β-lactam antibiotics but also resistant to aminoglycosides, quinolones, sulfonamides, and so on (Ho et al., 2007).
The epidemic situation, resistance and genotype distribution of ESBL-producing strains have varied with different countries, regions and even hospitals. ESBL strains have extended all over the world stage by stage, while detectable rates have shown significant difference in different hospitals or areas of the same country. Previous researches have indicated the detectable rate of ESBLproducing strains from 5 to 8% of Escherichia coli Isolates in Korea, Japan, Malaysia, and Singapore, from 12 to 24% in Thailand, Taiwan, Philippines, and Indonesia (Paterson and Bonomo, 2005), from 62 to 100% in India (Mathai et al., 2009). By contrast, the rate was almost up to 60% in China (Xiao et al., 2011).
Given few wide-ranging survey researches in Shandong province, China, we aimed to explore ESBLs epidemiology, resistance and resistant genotypes of 289 E. coli strains Isolated from three hospitals in that area. In addition, we have collected clinical data about the patients to carry on further research. The study helps us reveal their distributions and local constitutions in that area, thus providing guidance for clinical treatment, and then contributing to avoiding delay the conditions of patients and medical resources waste.

Bacterial Strains
We have collected 289 clinical Isolates of E. coli from patients in three typical hospitals of Shandong province in, China, from July 2013 to August 2014. There exist no replicate strains Isolated from one patient in our samples. Additional information has been collected with each Isolate, including isolation date, origin of the specimen, demographic details. Our experiment has been approved by Medical Ethics Committee of Shandong University School of Medicine (Grant No. 201401048).
The disk diffusion method was applied to assess ESBL production in all the Isolates with cefotaxime (30 ug) and ceftazidime (30 ug) alone and in combination with clavulanic acid (10 ug) as recommended by the Clinical and Laboratory Standards Institute (CLSI).

Nucleic Acid Extractions
DNA had been extracted from the Isolates by using boiling methods. Each strain was suspended in 500 ul of distilled water at concentration of MacFarland 0.5 and boiled at 100°C for 10 min. Then spined it in a centrifuge and kept the supernatant fluid in the −20°C freezer.

Polymerase Chain Reaction Detection and Sequencing of ESBLs
To determine the genotype of ESBLs, we performed polymerase chain reaction (PCR) amplification with the TEM and CTX-M. ESBLs Isolates were amplified with primers (Essack et al., 2001): 5 ′ -ATGAGTATTC AACATTTCCGTG-3 and 5 ′ -TTACCAATGCTTAATCAGTGAG-3, which have been designed to amplify TEM gene (length, 840 bp). Cycling conditions were as following: initial denaturation at 95°C for 3 min; 35 cycles of 95°C for 1 min, 55°C for 1 min and 72°C for 1 min; and a final elongation at 72°C for 5 min. We applied CTX-M gene primers: C1 (5 ′ -SCSATGTGCAGYACCAGTAA-3) and C2 (5 ′ -CCGCRAT ATGRTTGGTGGTG-3) to detect CTX-M gene (length, 554 bp). Cycling conditions were as follows: initial denaturation at 94°C for 3 min; 35 cycles of 94°C for 30S, 55°C for 30s and 72°C for 45s; and a final elongation at 72°C for 5 min. PCR reactions for each strain were repeated at least three times.

Sequencing of TEM and CTX-M genes
The PCR products were purified with a QIA quick PCR purification kit (Qiagen, Hilden, Germany) and sequenced on an ABI Prism 377 automated sequencer (Perkin-Elmer, Norwalk, CT, USA). The primers for sequencing were the amplification primer. All sequences were confirmed by two independent determinations and analyzed by Basic Local Alignment Search Tool.

Antimicrobial Susceptibility
The ESBL-producing Isolates were often multidrug-resistant. ESBL producers have shown much higher rates of resistance than those non-ESBL to ciprofloxacin (ESBL versus non-ESBL,

Characteristics of the ESBL-producing Isolates
All 197 Isolates with an ESBL phenotype tested positive for blaCTX-M and blaTEM using the consensus primers. We found 172 (87.3%) Isolates were positive for blaCTX-M, while 142(72.1%) Isolates were positive for blaTEM and 11 non-TEM-CTX-M-type Producers. In addition, most Isolates appeared to produce both TEM and CTX-M enzymes (128). Using nucleotide sequence analysis we found that the TEM enzymes detected two types which were TEM-1(138) and TEM-135(4) β-lactamase.

Discussion
In the article, 289 E. coli Isolates were collected from patients from July 2013 to August 2014; the detection rate of ESBL producers was 68.2% which was significantly higher than previous researches (Xiao et al., 2011). Therefore, we should pay more attention to the problem and try to take appropriate prevention and remedial measures. We found that much more ESBL-producing Isolates in urinary surgery ward and ICU and the detection rate were higher than the other wards. Because a majority of patients in ICU acquired serious diseases lowering their immunity, and involved heavy use of antibiotics for a long time, both of which contributing to higher detection rates. And for patients in urinary surgery, higher detection rates were related to their selves characteristics, one of which was mainly urinary tract obstruction in favors of bacteria propagation, in addition to urethral catheterization, further increasing opportunities for infection. Besides, we found most ESBL-producing strains occur in old man or woman, because the elderly themselves have low immunity. And in the patients, one patient over 60 repeatedly infected with the ESBLproducing E. coli. As a long duration of extended-spectrum βlactamase-producing Enterobacteriaceae carriage after discharge  (Birgand et al., 2013), we concluded that those patients underlying chronic illnesses which had lowered their immunity were apt to be infected with ESBL-producing E. coli. Therefore, we should initiate active interventions and treatments. The susceptibility test data showed that the ESBL producers which were resistant to most-lactams and frequently resistant to the non-β-lactam antibiotics, for example, fluoroquinolones and aminoglycosides. If patients are infected by ESBL producers, cefmetazole, or imipenem will be chosen before results of antibiotic susceptibility test having been done. But if patients are in a critical condition, we should make choice of carbapenems (Paterson, 2000). ESBL producers have shown much higher rates of resistance than those non-ESBL. Therefore, we should avoid antibiotics abuse in outpatient clinic and community hospitals, reducing opportunities for emergence of ESBLs.
TEM is the main type of β-lactamase, the TEM-1 group being the most common ones. CTX-M enzymes was a new group of plasmid-mediated ESBLs which has become the predominant ESBLs reported in Europe from last decade and has increased dramatically in many countries (Bonnet, 2004;Cantón and Coque, 2006;Livermore et al., 2007;Pitout and Laupland, 2008). In different geographic areas, the antibiotic consumption and dissimilar risk factors might have also contributed to the current epidemiology of CTX-M enzymes. Our study found that the CTX-M-14 and CTX-M-15 were in the majority, Dolejska et al. (2011) andHiroi et al. (2012) coincided with that.
Even though we have explored ESBLs epidemiology, resistance and resistant genotypes in three hospitals of Shandong province, with limitation of small samples, we cannot provide enough theoretical and practical guidance for clinical treatment. In the future research, we should base upon more large survey samples from perspective of increasing patients, hospitals and regions. On the other hand, because of much more ESBL producers resistant to carbapenems, to conduct a targeted research, we will focus on carbapenem-resistant Enterobacteriaceae (Centers for Disease Control and Prevention, 2013;Asakura et al., 2014). Given carbapenems being the first-line agent for patients with ESBL-producing bacteria, the occurrence of resistant organisms could be related to carbapenems, and then we will explore whether non-carbapenems plays more efficient role in infections with ESBL-producing Enterobacteriaceae.
Generally speaking, much more ESBL producers are detected among E. coli in China. The drug resistance rate of E. coli is high, with the tendency getting higher. Attaching more importance to monitor the drug resistance of bacterium and reasonable application and control abuse of antibiotics in clinic. Based on local database, our research have clarified the clinical and epidemiological features in Shandong province, which contributes to provide guidance for identifying appropriate treatment plans according to patients characteristics, especially for patients underlying chronic illnesses. We should take measures appropriately, and then contributing to avoiding delay the conditions of patients and medical resources waste.