Prevalence and characterization of extended spectrum beta-lactamase-producing Enterobacter cloacae strains in Algeria

Introduction: Expended spectrum β-lactamase (ESBL)-producing Enterobacter cloacae is an important nosocomial pathogen. In this study, the prevalence and the molecular epidemiology of ESBL producing E. cloacae strains isolated from various hospitals in Annaba, Algeria were investigated. Methodology: The study involved 63 isolates of E. cloacae obtained during 2009 at the four hospitals in Annaba. The detection of ESBL was performed using the double-disk synergy test and the combined disk test. Minimum inhibitory concentrations (MICs) were determined using the agar dilution method. The presence of blaCTX-M, blaSHV, blaTEM, and blaDHA β-lactamase genes was evaluated by PCR, and genomic typing was determined by pulsed-field gel electrophoresis (PFGE) analysis. The clinical and microbiological data were entered into the EpiI Info database. Results: Thirty isolates (47.6%) had an ESBL phenotype. BlaCTX-M group1 (76%); blaTEM (70%) were the most prevalent, followed by blaDHA (16.6%) and blaSHV (10%). Eighteen strains expressed at least two bla genes. MICs revealed a high level of resistance to cefotaxime, ceftazidime, and cefepime. PFGE revealed an epidemic clonal dissemination of these isolates. Various risk factors associated with the occurrence of ESBL-producing E. cloacae were detected. Conclusions: A higher frequency of ESBL-producing isolates and a diversity of β-lactamases were detected among ESBL-producing E. cloacae; these resulted from an epidemic clonal dissemination and high transference of ESBL genes between bacteria in hospital settings. Strict measures will be required to control the further spread of these pathogens in hospital settings.


Introduction
Infections caused by expended spectrum βlactamase (ESBL)-producing bacteria result in increased mortality in hospitalized patients [1].Invasive procedures, admission to intensive care units, and previous use of antibiotics are the most common risk factors for ESBL-producing bacteria [2].Enterobacter species, particularly E. cloacae and E. aerogenes, are important nosocomial pathogens responsible for various infections [3][4][5] and are therefore under intensive selective pressure from broad-spectrum β-lactam use [6].
Various kinds of β-lactamases have been described in E. cloacae.The chromosomally encoded cephalosporinase is common to all strains of E. cloacae.In addition to the class C cephalosporinase, other β-lactamases have been reported in this species, including ESBL enzymes [7].
These ESBL genes are known to spread to other members of the Enterobacteriaceae and have been classified into nine distinct structural and evolutionary families based on their amino acid sequences [8].TEM and SHV enzymes form major families.In the past decade, CTX-M type has emerged in many countries [9].Currently, the CTX-M family includes more than 130 β-lactamases, which are grouped on the basis of sequence similarity into five distinct clusters (subtypes): CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25 (www.lahey.org/Studies/).
The present study explored the prevalence and molecular epidemiology of ESBL-producing E. cloacae strains isolated from various hospitals in Annaba, Algeria.An attempt was also made to detect the resistance of ESBL strains to other antimicrobial groups and to analyze various risk factors associated with the occurrence of infections by ESBL-producing strains.

Preparation of DNA template for PCR
Total DNA was extracted by suspending a few colonies of overnight culture of E. cloacae isolates growing on Luria Bertani agar (Bio-Rad, Marnes-la-Coquette, France) in 500 µL of DNase-and RNasefree water (Invitrogen, Paisley, UK).The suspension was boiled at 100°C for 10 minutes in thermal block (Polystat 5, Bioblock Scientific, France), then centrifuged at 19000 x g for 5 minutes.An aliquot of 1 µL of the supernatant was used as the DNA template for PCR.

Detection of β-lactamase-encoding genes
E. cloacae isolates were screened by PCR for the following β-lactamase encoding genes: bla CTX-M and bla DHA as described previously [12].All primers used in this work are shown in Table 1.Amplification reactions were performed in a volume of 50 μL containing 2 μL of DNA template, 2.5mM MgCl 2 , 0.4 μM of each forward and reverse primers, 100 μM of each dNTP, and 2 units Taq DNA polymerase (Promega, Madison, USA) in 1 × PCR buffer provided by the manufacturer.
Cycling parameters included five minutes of denaturation at 95°C, followed by 30 cycles of denaturation (95°C for one minute), annealing (60°C for one minute for CTX-M and SHV, 52°C for one minute for TEM, 64°C for one minute for DHA), and extension (72°C for one minute), ending with a final extension period of 72°C for seven minutes.
PCR products were detected on 1.5% agarose gel (FMC Bioproduct, Rockland, USA) after ethidium bromide staining and UV illumination, photographed with an Olympus digital camera, and analyzed using Digi-Doc-it software (UVP, Upland, USA).
Genetic relationships Genetic relationships of the ESBL-producing E. cloacae were assessed using PFGE after digestion with XbaI.The PFGE analysis of XbaI-digested genomic DNA was performed using a CHEF-DRIII apparatus (Bio-Rad, Hercules, USA) according to the instruction manual.Bacterial DNA and XbaI digestion was prepared as previously described [13].Fragment separation was done in 1% agarose gel with the following conditions: 6 V/cM for 22 hours with switch times of 5-30 seconds in Tris-borate-EDTA buffer prechilled to 12°C.
The similarity of the PFGE banding patterns was analyzed by the Dice coefficient, and the data obtained were analyzed by the unweighted pair group method with arithmetic average (UPGMA) clustering using the Pearson correlation coefficient (Biochemistry and Biotechnology Department, Rovira i Virgili University, Tarragona, Spain) (http://genomes.urv.cat/UPGMA/index.php).
Interpretation of chromosomal DNA restriction patterns was based on the criteria of Tenover et al. [14].Briefly, strains showing more than three DNA fragment variations and a similarity of < 80% at dendrogram analysis were considered to represent different PFGE types, while one-to three-fragment differences and a similarity of > 80% upon dendrogram analysis were considered to represent PFGE pattern subtypes.

Statistical analysis
Several epidemiological data were sought systematically in the clinical records of patients infected with ESBL-producing E. cloacae: age, sex, wards, site of sampling, nature of infection, length of stay, first prescription of antibiotics, history of hospitalization in the last year, mode of admission (direct or transfer), field of patient and often underlying pathology, and number of invasive procedures.A descriptive analysis using Epi Info version 6 software (CDC, Atlanta, USA) was performed.All data were recorded using Excel (Microsoft, New York, USA), and the statistical analysis was performed using Epi Info version 6 software.

Results
A total of 63 patients were included in this study.Thirty (47.6%) patients were enrolled as ESBLproducing E. cloacae strains, and 33 (52.4%) patients were included as non-ESBL.
The median age was 41 years (range = 9 days to 73 years), and 63.3% were male.The average length of hospitalization was 13.2 days (range = 6 days to 29 days).Patient characteristics and laboratory data are described in Table 2.
The total DNA isolated from 30 ESBL-producing organisms were subjected to PCR using TEM, SHV, and CTX-M specific primers.Two or more genes for ESBL were present in 15 (50%) of 30 ESBL typeable isolates, bla TEM + bla CTX-M, being the most common combination (33.3%) followed by bla TEM + bla SHV + bla CTX-M (6.6%), and bla TEM + bla SHV (3.3%).Among the isolates harbouring a single ESBL gene, bla CTX-M was present in 30 per cent and bla TEM in 20 per cent.All CTX-M enzymes in E. cloacae belonged to the CTXM-1 cluster.
The cephalosporinase DHA type was found in five E. cloacae.This gene has been presented with the following combinations: bla DHA-1 + bla CTX-M (n = 3) and bla DHA + bla CTX-M + bla TEM (n = 2).PFGE analysis revealed fourteen different clonal types (Figure 1) among the 30 ESBL-producing E. cloacae isolates.Eight of these clones were represented by a single isolate each (isolate E3, clone CE3; isolate E17, clone CE7; isolate E29, clone CE8; isolate E2, clone CE9; isolate E10, clone CE10; isolate E13, clone CE11; isolate E28, clone CE12, and isolate E14, clone CE14).The other six clones were represented by six (clone CE2), five (clone CE1), four (clone CE5), three (clone CE4) and two (CE6 and CE13) isolates, respectively (Table 2).Clone CE5 and CE1 were isolated from two different hospitals.In contrast, the same isolates of other clones were found in patients hospitalized in the same ward within a few months of each other, showing that an undetected limited outbreak of ESBL-producing E. cloacae had occurred in that unit.
Various risk factors associated with the occurrence of ESBL-producing E.cloacae are shown in Table 2.When these factors were further subjected to multivariate analysis and antibiotic exposure (therapy), 17 of the 23 cases identified during the study period had been given a presumptive treatment of third-generation cephalosporins antibiotic, either alone (4/17) or combined with aminoglycoside (11/17) and fluroquinolone (2/17).Patients' use of a urinary catheter (70%) was found to be significantly associated with infections by ESBL-producing isolates.

Discussion
E. cloacae is frequently responsible for nosocomial infections, including urinary tract infections, pneumonia, and bloodstream infections [15][16].In the current study, infections caused by ESBL-producing E. cloacae isolates were mainly urinary infections, followed by surgical wound infections and pulmonary infections.Theseresults are in accordance with the results reported in the literature [17].
This study found that the prevalence of ESBLproducing E. cloacae isolates in the Annaba hospitals was 47.6%, which was higher than that the prevalence rates found in Algerian hospitals (17.7%) [18] and in Korea (35.4%) [19].E. cloacae with ESBL production has been reported to exceed 43% in countries such as Singapore, mainland China, and the Philippines [20].The use of cefotaxime and, recently, ceftazidime in Algeria could partly explain this high percentage of ESBLs.Overall, the resistance patterns of ESBL- producing E. cloacae studied here were similar to those commonly described in other studies (i.e., the ESBL producers were resistant to different antibiotic families including β-lactams, fluoroquinolones, aminoglycosides, and trimethoprim/sulfamethoxazole) [18,[21][22][23].
The CTX-M type was the most common ESBL in the Annaba hospitals, which was higher than that reported from Algerian hospitals (20%) [18].CTX-M group 1 remains the only subgroup of CTX-M ESBLs found in Algeria with the predominance of CTX-M-3 and CTX-M-15 enzymes [18,24].Encoding genes for the SHV family were detected in 10% of the isolates.Most enzymes from this group are ESBL, except for SHV-1 and SHV-11 [25][26].However, the enzymes from this group cannot be confirmed to be ESBLencoding genes, mostly in strains presenting more than one bla gene.On the other hand, the bla SHV-12 gene was also detected in E. cloacae from Algerian hospitals [21].The bla TEM gene was identified in 70% of isolates, generally in association with SHV and/or CTX-M genes.Therefore, it was not possible to determine if they were responsible for the ESBL phenotype or not; they could be non-ESBL encoding genes associated with another gene expressing the extended-spectrum phenotype [27].On the other hand, co-production of different ESBLs by a unique strain has been a common finding in several studies [18,[25][26]28], so the carriage of two or three ESBL-encoding genes by the TEM-producing strains of this study could also have occurred.
In the present study, PFGE revealed fourteen different pulsotypes among the 30 ESBL-producing E. cloacae isolates.The services most affected by the clonal spread were infectious disease (6 clones), surgery (5 clones), and paediatrics wards (4 clones).These wards have patients with fragile host defences (36% of patients harboured a disease) receiving excessive manipulation and a high antimicrobial intake (74% of patients had received antibiotic treatment), which could have been predisposing factors for infections and the spread of resistance genes.A total of 46% of patients in the current study were already hospitalized in other wards and other hospitals; the movements of patients between different hospital wards must therefore also be considered.
In the univariate analysis, prolonged hospital stay, intensive care unit admission, urinary catheter use, and elective surgery were risk factors associated with ESBL strains.All these conditions are related to the severe conditions of the patients, as well as the use of broad-spectrum antibiotics.A previously published study verified irrelevant risk factors for Enterobacter bacteremia among other bacteria, but no comparison of different resistance profiles was made [29][30].Chang et al. [31] showed that fourth-generation cephalosporin resistance was an independent risk factor of Enterobacter bacteremia.Prior antibiotic administration is a major factor for colonization and secondary infections with these multiple-antibioticresistant organisms.Clinicians are advised to avoid unnecessary administration of antimicrobial agents and to avoid unnecessary prolonged administration.For surgical prophylaxis, administration of antibiotics for longer than 24 hours is rarely justifiable.Education programs for hospital personnel about risk reduction in the transmission of Enterobacter species and other nosocomial pathogens should be implemented in hospitals.This is usually the responsibility of the infection control team.

Conclusion
In summary, the data obtained in this study showed that higher rates of ESBL-producing E. cloacae isolate in Annaba hospitals have resulted from an epidemic clonal dissemination in different hospital wards.Various risk factors associated with the occurrence of ESBL-producing E. cloacae were detected.Genes bla CTX-M and bla TEM were detected in high frequency among ESBL-positive isolates, occurring alone or in combination, indicating the high transference of ESBL genes between bacteria in hospital settings.Strict measures will be required to control the further spread of these pathogens in hospital settings.

Figure 1 .
Figure 1.Dendrogram and PFGE of XbaI-digested genomic DNAs from ESBL-producing E. cloacae collected in Annaba hospitals of Algeria

Table 1 .
Primers used for detection of SHV, CTX-M, TEM, and DHA genes

Table 2 .
Characteristics of patients infected with ESBL-producing E. cloacae

Table 3 .
Characteristics of ESBL-producing E. cloacae detected in Annaba hospitals