Nosocomial infections: Importance of rapid and early detection of ESBL Enterobacteriaceae by molecular biology

Nosocomial infections are a major public health problem. They are mainly caused by bacteria that often present antibiotic resistance profiles, which complicates their management. The diagnosis of these infections is based on clinical and biological criteria that lack sensitivity and specificity, and on microbiological examinations that are lengthy. Unfortunately, the inadequate empirical antibiotic therapy in many cases, and the late implementation of an effective treatment, are associated with the emergence and spread of bacteria that are multi-resistant to antibiotics, i.e. strains producing extended-spectrum beta-lactamases (ESBL). Rapid diagnostic methods, identifying the pathogen and its resistance profile, are therefore necessary. Our laboratory is committed to develop PCR techniques to genotypically identify the most frequent ESBLs from four hospital facilities. Among all the enterobacteria detected, we observed the predominance of OXA type ESBL (38%), followed by CTX-M type ESBL (33%). TEM and SHV genes represent respectively 19% and 10%.The optimization of these technologies could allow the identification of all known resistance mechanisms in only a few hours and find new preventive or curative strategies in the fight against these nosocomial infections, to better respond to this health threat.


Introduction
The production of beta-lactamases is the main cause of resistance to beta-lactamase antibiotics in gram-negative bacteria. These enzymes cut the amide bond in the betalactamase cycle, rendering beta-lactamase antibiotics harmless to bacteria [1]. There are many types of ESBLs such as TEM, VHS, CTX-M, OXA, AmpC, etc., but TEM and sulphydrylvariable VHS are the major types of ESBLs and these enzymes are most commonly found in E. coli and K. pneumoniae [2]. Determination of TEM and VHS genes by molecular techniques in ESBL-producing bacteria and their antimicrobial resistance profile can provide useful data on their epidemiology and risk factors associated with these infections [4].
The genes encoding these enzymes are located on transferable plasmids [5]. CTX-M type betalactamases are a new group of enzymes encoded by transferable plasmids [6]. The name CTX-M type Betalactamases is due to their high activity against cefotaxime. Unlike TEM and SHV ESBLs, most CTX-M enzymes preferentially hydrolyze and confer resistance to cefotaxime and ceftriaxone rather than ceftazidime. In recent years, a new family of plasmid-

Amplification
The amplifications were performed under a volume of 52 μl (including 50 μl of reaction mixture and 2 μl of DNA extract) using the thermocycler (PROGEN), programmed. The different steps of the amplification, the temperature and the time to perform each step are mentioned in the ( Table 2).

Results
In 2014, 37 strains of enterobacteria were isolated from four Moroccan regions to study their resistance profile.
Among these 37 isolates, 22 indicate the profile of ESBL enterobacteria. During this period, the most ESBL-producing bacterial species were Escherichia coli (n= 11), Klebsiella pneumoniae (n= 8), Enterobacter cloacae (n= 2) and Morganella Morganii (n= 1) (Fig. 1). These 22 ESBL-producing isolates were selected for detection of CTX-M, SHV, TEM and OXA genes by conventional PCR.  When we studied the different profiles existing in our strains, we had 8 different profiles presented in (fig 3).

Discussion
The results of our study confirm the high prevalence of the OXA and CTX-M genes, while the prevalence of the other TEM and SHV genes was moderately low. The majority of strains had two or more ESBL genes, and only two strains carried all four ESBL gene types. Our isolates came from a variety of Moroccan hospitals, suggesting that these beta-lactamases are widely distributed. ESBL production is much lower in Europe than in Latin America and Asia, and even lower in the Pacific than in North America. CTX-M ESBLs are easily recognized in antibiotic susceptibility testing, as CTX is the most affected molecule with very good inhibition around the tazobactam-containing disc. These primers have been used to amplify all strains with this profile [8].
In our study, 16 ESBL strains among the 22 expressed the CTX-M gene; Escherichia coli being the most common (7 strains), followed by Klebsiella pneumoniae (6 strains) and finally Enterobacter cloacae (3 strains). It appears clearly that the CTX-M gene predominates in our ESBL strains. The same result was found in Europe, while in other countries the ESBL genes are more diversified [9]. ESBLs were inhibited by clavulanic acid and tazobactam. Importantly, the isolates were more resistant to cefotaxime and aztreonam than to ceftazidime, suggesting that they were CTX-M producers. Some CTX-M ESBLs confer high resistance to ceftazidime [10].

As elsewhere in Europe, the rate of CTX-M genes is increasing with the new century, especially in northern
France [9]. In recent years, the appearance of new variants of ESBL producers, in particular CTX-M, has raised the implication of co-resistance to other drug classes in endemic situations. This co-resistance is due to the propagation of different types of resistance genes within the same clone. Some works have reported that bla CTX-M genes are frequently located on large plasmids that often carry other genes attributing resistance to other antimicrobial agents, citing aminoglycosides, fluoroquinolones, chloramphenicols, tetracyclines, and others… This may explain the high rate of spread of the CTX-M gene among E. coli strains through the acquisition of the R plasmid, and constantly the high prevalence of the CTX-M resistance gene is combined with another resistance gene in these strains [12]. Recent European studies on enterobacterial resistance suggest that, EMT and VHS now replace as the predominant ESBL isolate. The prevalence of ESBL production has shown remarkable geographical differences, ranging from 0% in (Iceland) to less than 1% for (Estonia) and from 41% for E. coli and 91% for K. pneumonia in (Romania). A recent study by Lal et al describes the occurrence of genetic variants in K. pneumoniae from clinical specimens of diverse origins. This study reported that isolates with both TEM and SHV genes were more frequent than TEM and SHV alone [3].
In a study that was conducted in Iran, Amir Peymani et al. found that 58.5% of ESBL-producing Enterobacter cloacae isolates carried OXA-1 genes [13]. Unfortunately, very few epidemiological studies have been conducted to assess the spread of OXA ESBLs [14]. Several studies have been cited that address the resistance and frequency of ESBL in Asia and specifically in India. This may be due to a number of factors that may give rise to this problem; the lack of sewage (the "Delhi belly") and poor quality of drinking water, coupled with a lack of control over the prescription and sale of antibiotics, are probably major factors that have facilitated the spread of resistance [15]. rates for TEM, SHV, and CTX-M beta-lactamases, respectively. While, there were no strains harboring OXA type beta-lactamase in their study [4]. From Portugal, Soraia Necho Amaral reported that the genes bla TEM and bla OXA were found in those isolates representing by a number of (8/12 and 9/12, respectively) [17].
Another study summarized that bla TEM was present in 63.3% (55/87) and bla SHV in 52% (45/87) isolates alone or in combination. OXA-type beta-lactamases were present in combination with other enzymes. It was present in 26.4% (23/87) isolates only in combination with other genes [18]. Given the epidemiological importance of β-lactamase genes, all families should be studied in Europe, especially the OXA and PER enzyme families that have been prevalent in other sites in Turkey [21].
Another study showed that transfer of genotypically related ESBLs from hospital to hospital within a single city, from city to city and from country to country. Intercontinental transfer has also been reported. VHStype ESBLs may be more common in clinical isolates than any other ESBL type.
The evolution of OXA-type β-lactamases from related enzymes with narrower spectra has many parallels with the evolution of SHV-and TEM-type ESBLs [22].
The epidemiology of ESBL is quite complicated. Initially, there are certain factors that are involved: the wider geographical area, the country, the hospital, the community and the host. In addition, there are bacteria and their mobile genetic elements, usually plasmids. In addition, there are several reservoirs, including the environment, wild animals, farm animals and pets [17].

Conclusion
Correct detection of ESBL-producing microorganisms is a challenge for laboratories, as it requires not only phenotypic tests, but also genotypic tests for all genes associated with beta-lactamase production. The prevalence of ESBL-producing bacteria is increasing every year, especially in tertiary hospitals.