Characterization of carbapenemase-producing Gram-negative bacilli: first report of blaNDM - 1 in Enterobacter cloacae

Introduction: The spread of multidrug-resistant bacteria, particularly carbapenem-resistant Gram-negative bacilli (CR-GNB), has become a serious challenge for clinicians due to limited therapeutic options. The aim of the study was to investigate the prevalence of carbapenemase production among clinical isolates recovered from 352 samples collected in Tebessa hospital, Algeria. Methodology: Bacterial isolates were identified by 16S RNA gene sequencing and susceptibility to antibiotics was determined by disk diffusion method. Carbapenem-resistant isolates were screened for carbapenemase production using modified carba Nordmann-Poirel test, modified Hodge test and imipenem-EDTA combined disc test. Extended-spectrum β -lactamases (ESBL) were detected using double-disk synergy test. Molecular characterization of carbapenemases and ESBL genes was performed by polymerase chain reaction (PCR) and sequencing. Results: A total of 85 Gram-negative bacilli isolates were recovered mainly from urine samples and were identified as: Klebsiella pneumoniae (17.65%), Serratia odorifera (15.29%), Escherichia coli (12.94%), Raoultella ornithinolytica , Enterobacter cloacae (11.76%), Serratia marcescens (10.59%), Morganella morganii (7.06%), Proteus mirabilis (5.88%), Acinetobacter baumannii (4.70%) and Pseudomonas aeruginosa (2.35%). All strains were resistant or intermediate to imipenem and/or ertapenem. ESBL, carbapenemase and metallo-beta-lactamases (MBL) phenotypes were detected in 19 (22.35%), 9 (10.59%) and 2 (2.35%) GNB isolates, respectively. PCR results in nine carbapenemase-producing GNB strains chosen showed the presence of one to four carbapenemase genes (bla GES , bla SME , bla NDM-1 , bla VIM , bla GIM , bla SPM , bla OXA-48 ) in four strains; however, seven strains had at least one ESBL gene ( bla TEM-1 , bla CTXM-15 , bla SHV ). Conclusions: In this study, we report the first incidence of bla NDM-1 gene in Enterobacter cloacae isolated from urine sample in Algeria.


Introduction
Carbapenems are a class of antimicrobial agents used most frequently as last resort antibiotics for the treatment of infections with multidrug-resistant (MDR) Gram-negative bacilli (GNB), since they have the wide spectrum of bactericidal action and stability against most of the β-lactamases including ESBLs [1,2].However, with the extensive use of these antibiotics, the number of carbapenem-resistant Enterobacteriaceae (CRE) are emerging and increasing rapidly [3].Carbapenemase is the main determinant contributing to carbapenem resistance in Enterobacteriaceae.Indeed, the widespread use of these antibiotics has caused the expansion of resistant Enterobacteriaceae [4].The concern about carbapenemase-producing Gramnegative bacilli (CP-GNB) that has now emerged is that it is often associated with the occurrence of MDR isolates for which there are few antimicrobial options available [5].Carbapenemases are β-lactamases that hydrolyze carbapenems (imipenem, meropenem, and ertapenem).These β-lactamases are now extensively identified in GNB, particularly in Enterobacteriaceae [6].
Various carbapenemases have been reported in Enterobacteriaceae including Enterobacter, Klebsiella, Escherichia coli, Serratia [7] and other opportunistic GNB such as Acinetobacter and Pseudomonas [8].Nevertheless, carbapenem resistance in GNB may be due to other drug resistance mechanisms such as modification or loss of porins and efflux pumps [9].
Among the recent spread of multidrug-resistant bacteria, outbreaks of ESBLs and carbapenemaseproducing GNB are a serious problem not only making treatment difficult but also worsening the prognosis of infected patients.In this background, the objective of our study is to assess the prevalence of antibiotic resistance and to characterize carbapenemaseproducing strains among clinical GNB isolates collected in Tebessa hospital, Algeria, using phenotypic (modified Hodge test, Carba Nordmann-Poirel NP test, imipenem-EDTA combined disc test) and molecular (polymerase chain reaction -PCR and sequencing) tests.

Study setting and bacterial isolates
A total of 85 non-redundant GNB strains were isolated during the period between February and May 2018, at Tebessa hospital, from 352 clinical specimens including: urine (n = 281), pus (n = 28), dialysis fluid (n = 21), blood culture (n = 13) and cerebrospinal fluid (n = 9).Approximately 15000 patients were admitted at the outpatient department per year and more than 900 operations and invasive diagnostic therapeutic procedures were performed annually in this hospital.Samples taken from patients hospitalized in various services and from outpatients were cultured on Mac Conkey agar and cetrimide agar (Fluka, La Chapellesur-Erdre, Cedex, France).GNB identification was based on colony morphology and biochemical characteristics using API 20E, a semi-automatized assay (bioMérieux, Marcy l'Etoile, France).Isolates were frozen at -30 °C in brain-heart infusion broth with 15% glycerol until processed for further experimentation.

Antibiotic susceptibility testing
Antibiotic susceptibility testing of Enterobacteriaceae and non-fermentative GNB (NF-GNB) isolates was performed on Mueller-Hinton agar (BioMérieux, Marcy-l'Étoile, France) by standard disk diffusion method, using disk antibiotics (Liofilchem, Roseto degli Abruzzi TE, Italy) according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [13].Klebsiella pneumoniae ATCC700603 and Escherichia coli ATCC25922 strains were used as controls.The isolates were defined as MDR when they were resistant to at least three antibiotics from different classes [14].The minimal inhibitory concentrations (MICs) of antibiotics were determined by the microdilution method recommended by EUCAST [13].

Phenotypic carbapenemase detection
Isolates with decreased susceptibility (intermediate/resistant) to at least one of the carbapenems should be considered as carbapenemresistant (CR) and suspicious for carbapenemaseproducing (CP).Thus, isolates with a reduced sensitivity to ertapenem or to imipenem were screened for carbapenemase-producing strains by the Carba NP test and the modified Hodge test [13].

Modified Hodge test (MHT)
CR isolates were subjected to HT as was described in the CLSI guidelines [16].Mueller-Hinton agar (MHA) plates were inoculated with an overnight culture of E. coli ATCC 25922 adjusted to one tenth turbidity of 0.5 McFarland.The plates were left for 15 minutes to dry and then ertapenem disc (10 μg) was placed at the center of the plate.Using a swab, overnight cultures of the tested isolates (3-5 colonies) were streaked from the edge of disc to the periphery of the plates and the plates were incubated at 37 °C for 24h.Carbapenemase-producer isolates were indicated by enhanced growth of E. coli around tested isolate, expressed as clover leaf like indentation, while no enhanced growth of E. coli indicates a non carbapenemase-producing isolate.

Carba NP test
The Carba NP test was performed following the protocol recommended by CLSI [16].Briefly, bacteria were cultured overnight on MHA and then the bacterial mass was scraped off with a 1μL loop and suspended in a 1.5 mL Eppendorf tube containing 100 μL of 20mM Tris-HCl lysis buffer and mixed using a vortex device for 5 seconds.This lysate was mixed with 100 μL of an aqueous indicator solution consisting of 0.05% phenol red with 0.1 mmol/L ZnSO 4, previously adjusted to pH 7.8 and 6 mg/mL imipenem (reaction tube) and, as a control tube, the phenol red solution without antibiotic.Tubes were vigorously mixed for 5 to 10 seconds before incubation.Finally, tubes were incubated at 37 °C and monitored for 2 hours for colour change from red to orange/yellow in the tube containing antibiotic, which was interpreted as a positive result.

Imipenem-EDTA combined disc test
This test is used to detect carbapenemases class B (MBL) which are inhibited by EDTA.It was carried out for Carba NP test (+) and/or MHT (+) strains.Imipenem-EDTA combined disc test was conducted according to Prakash et al. [17].The tested isolate was inoculated by swab on MHA, then a 10 μg imipenem (IPM) disk was placed on the plate at a distance of 20 mm from an Imipenem-EDTA (EIP) disk and the plates were incubated overnight at 37 °C.After incubation, the zone of inhibition of the imipenem and the imipenem-EDTA disks were compared.If the increase in the zone of inhibition with EIP disk exceeded 7 mm than IPM disk, the isolate was considered to be MBL positive.

Molecular identification of isolates
Bacterial isolates were grown in Luria-Bertani medium for 24 h at 30 °C.Thereafter, the genomic DNA was extracted as described by Wang et al. [24].The 16S rRNA amplification was carried out using primers 27F and 1492R.PCR amplifications were carried out in a final volume of 50 μL, containing 25 μL of 2 × Taq PCR Master Mix (TransGen Biotech, Beijing, CHINA), 2 μL of each primer (10 μmol L −1 ), 2 μL of template DNA (10 ng μL −1 ), and 19 μL of ddH2O.The cycling conditions were optimized with an initial denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 1 min, and 72 °C for 1 min; and a final extension at 72 °C for 10 min.The amplicons were sequenced and the gene sequences obtained were analysed and blast-searched in the GenBank database (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Bacterial isolates
In the present study, a total of 85 GNB with different colony morphologies were recovered from 352 clinical specimens including urine (63), dialysis fluid (11), pus (5), blood culture (3) and cerebrospinal fluid (3).Of these, 79 (92.94%) isolates were identified as members of Enterobacteriaceae family and 6 (7.06%) isolates were identified as non-fermentative Gram-negative bacilli (NF-GNB).The distribution of the isolates from various samples and wards is presented in Table 1.
The distribution of isolates from various clinical specimens showed that urine was the source of 63/85 (74.12%) of the isolates.Dialysis fluid (12.94%) was the second major source of isolates reflecting the relatively high frequency of GNB involved in bacteremia.The remaining isolates collected in our study were from pus, blood culture and cerebrospinal fluid.

Antibiotic susceptibility and phenotypic characterization
Antimicrobial resistance patterns of GNB isolates are summarized in Table 2.The results showed that most of the isolates were resistant to amoxicillin, amoxicillin/clavulanic acid, ticarcillin, cephalexin and cotrimoxazol.The isolates had a high resistance rate to fluoroquinolones (ciprofloxacin), exceeding 64% for K. pneumoniae, R. ornithinolytica, E. cloacae and A. baumannii.In addition, the carbapenem resistance rate showed a worrying trend with 49/85 isolates (57.65%) for ertapenem and 68/85 isolates (80%) for imipenem.Besides, variable rates of resistance were noted for other antibiotics.Our results showed also that amikacin was the most active antibiotic with 80/85 sensitive strains (94.12%) for this antibiotic.Among the 85 GNB isolated, 58 (68.23%) strains exhibited MDR patterns to different classes of antibiotics.
The DDST results showed that 19/85 isolates (22.35%) were ESBL producers (Table 3).In the present study, all strains showed decreased sensitivity to at least one of the carbapenems tested (imipenem or ertapenem).Therefore, they were all screened for carbapenemase production by the Carba NP test and MHT.Results showed that Carba NP test was positive for 9/85 isolates (10.59%) and MHT was positive for 7/85 isolates (8.23%).The imipenem-EDTA combined disc test was positive for only two carbapenem-resistant strains (Figure 1), which indicates the presence of metallo-β-lactamase production in these strains.

Molecular characterization of ESBL and carbapenemase genes
In this study, 9 strains were detected as CP-GNB (Table 4) and they were subjected to molecular characterization for resistance genes.PCR for ESBL genes showed that seven strains contained bla TEM ± blaCTX-M genes: S. marcescens (two strains), E. cloacae (two strains) and A. baumannii (three strains) (Figure 2).In addition, blaSHV was detected in three strains: A. baumannii (two strains) and E. cloacae (one strain).Sequencing analysis of resistance genes encoding ESBL producers revealed that these genes were blaTEM-1, blaCTXM-15 and blaSHV.Moreover, the blaSHV and  blaCTX-M-15 combination was detected only in one strain identified as E. cloacae.
The CR-GNB strains were further tested to check if they carried carbapenemase genes.The PCR results (Table 4) showed that 4 out of 9 CP-GNB strains harbored at least one of the carbapenemase genes tested in our investigation.The genes detected were: blaGES (one strain of S. odorifera) and blaSME (two strains of E. cloacae) encoding class A carbapenemases; blaVIM and blaNDM (one strain of E. cloacae and one strain of A. baumannii) (Figure 2), blaGIM and blaSPM (one strain of A. baumannii) encoding class B carbapenemases (MBL); blaOXA-48 (one strain of E. cloacae) encoding class D carbapenemases (Figure 2).Interestingly, we have noted that one strain of E. cloacae and one strain of A. baumannii harbored more than three types of carbapenemase genes.

Discussion
Carbapenems are generally considered the most effective antibacterial agents for the treatment of multidrug-resistant bacterial infections.Unfortunately, with the widespread use of these antibiotics, CRE have increased dramatically over the last few years, and have been reported in many countries [2,25].
The present study investigated antibiotic susceptibility patterns and characterized ESBL and carbapenemase genes among isolates of GNB recovered from different clinical samples.Antibiotic susceptibility testing showed that out of 85 GNB isolated from urine, 58 (68.23%) were found to be MDR.This finding is higher than those reported at Annaba hospital -Algeria (35.53%) [26] and at Guelma hospitals (29%) [27].The high prevalence of MDR-GNB reported worldwide may be to higher selection pressure due to self-medication, misuse or overuse of carbapenems and third generation cephalosporins, frequent use of invasive devices and prolonged hospitalization [8].
Over the past decade, global data on ESBLproducing Enterobacteriaceae (ESBL-E) in North African countries have become extremely worrying and this region may well be one of the main epicenters of the global pandemic of ESBL.In the present study, the prevalence of ESBL-producing GNB was 22.35%, which is almost similar to that found in some North  African countries.In Algeria, data indicate a high prevalence of ESBL-E, where their prevalence has increased from 43.73% in 2014 to 44.16 % in 2015 [26].In recent study from Egypt, ESBL-E prevalence among clinical strains varied between 38.8% and 70.6% both in hospital and community acquired urinary tract infections, and the most frequently detected betalactamase genes were blaCTX-M and blaTEM [28].In Tunisia, the presence of ESBL-E has been increasingly reported, and their prevalence ranged from 11.7 to 77.8% causing both community acquired and hospital acquired infections [29].In Morocco, low prevalence rates between 1.3 and 7.5% have been found, of which blaCTX-M gene was the most prevalent [30].A survey conducted in Libya showed a prevalence of ESBL-E between 6.7 and 32.6% in hospital samples and 13.4% in community samples [31].
Furthermore, the emergence of ertapenem or imipenem-resistant GNB has become a universal concern leaving very few therapeutic options, as these molecules have been considered as preferred agents for the treatment of patients with moderate-to-severe infections and risk factors for infection with multidrugresistant GNB [26,32].The prevalence of carbapenemase-producing GNB strains in our study was 10.59% which is comparable with previous reports in Algeria [33].Due to the massive spread of CP GNB with overwhelming consequences in health care settings, the illogical and very common use of carbapenems has resulted in the emergence of CR-GNB strains [34].
Molecular identification of β-lactamases is considered to be a substantial trait for a reliable epidemiological investigation of antimicrobial resistance.Three types of ESBL were detected in our study: CTX-M-15, SHV and TEM-1.These findings corroborated a recent study [12] reporting ESBL-E coharboring extended-spectrum β-lactamase genes in community-acquired urinary tract infection.The emergence of the bla CTX-M-15 variant has been revealed to be attributed to the horizontal gene transfer of genetic elements and the clonal expansion of microorganisms [30].Furthermore, the widespread and unnecessary use of ceftriaxone and cefotaxime has led to the emergence and spread of blaCTX-M resistance genes [25].In concordance with previous reports, the co-existence of two or three different ESBL genes was detected in six of our isolates (Table 4).However, the most common combination of ESBL genes was between blaTEM, blaSHV, blaOXA and blaCTX-M, which was consistent with studies from Morocco [30] and Tunisia [35].
In our study, the blaOXA-48 and blaNDM-1 genes have been detected in one strain identified as E. cloacae.To the best of our knowledge, this is the first report on potential E. cloacae isolates co-harbouring carbapenem-resistance genes blaNDM-1 and blaOXA-48 in Algeria.In agreement with our results, the coharbouring of carbapenemase genes has been reported in several studies [12,34].It should be noted that the coexistence of these carbapenemase genes is a therapeutic challenge to clinicians, due to restricted treatment options and the potential for global spread by horizontal transfer [36].
In Algerian hospitals, the OXA-48 carbapenemase gene was the most important mediator of carbapenem resistance in Enterobacteriaceae [37,38].Therefore, few studies on OXA-48-producing E. cloacae have been reported in Algeria, the first one was reported in Guelma Hospital (Eastern Algeria) [27].It has since been widely identified in different Algerian regions [37,39].
In this investigation, we also reported the isolation of NDM-1-producing Enterobacter cloacae.This species is an important nosocomial pathogen, which can cause various infections including urinary tract, respiratory tract, surgical site, biliary tract, sepsis, intravenous catheters, central nervous system and outbreaks in neonatal units [3].E. cloacae carrying blaNDM-1 is extremely rare and, to our knowledge, has not been described in Algeria to date.Moreover, NDM-1-positive E. cloacae has been found in India [48], China [3], South Africa [6] and Japan [49], indicating that the blaNDM-1 gene has spread from K. pneumoniae to E. cloacae.Furthermore, blaNDM-1 has been also reported in other Enterobacteriaceae such as Raoultella ornithinolytica [50] and Escherichia coli [51].
The widespread dissemination of blaNDM-1 is mainly due to plasmids, integrons, insertion sequence common region (ISCR) and clonal outbreaks [52].The blaNDM-1 gene is mainly located on incompatible conjugative plasmids and can be transmitted among different bacterial species, resulting in extensive drug resistance [10].So far, most studies of NDM-1-positive isolates have focused on resistance mechanisms and transmission.However, it is not known whether blaNDM-1 affects the biological characteristics of the strains, such as growth ability and competitiveness.
Carbapenem-resistant GNB including Enterobacteriaceae are a matter of national and international concern because of their high levels of antimicrobial resistance and their association with high mortality.This study was not based on systematic surveillance of carbapenem resistance and so the sample size was small.However, our contribution can form the basis for further epidemiological studies of carbapenem-resistant GNB in order to promote appropriate antimicrobial therapy as well as to establish adequate infection control precautions and to stop the spread of these resistant bacteria.

Conclusions
This study reports for the first time the carbapenemase genes in Tebessa (eastern Algeria) hospital.It reports particularly the first incidence of bla NDM-1 in Enterobacter cloacae in Algeria.The detection of carbapenem-resistant and ESBL-producing GNB is of great concern and may significantly limit the efficacy of therapeutic options in hospital settings.There is thus an urgent need to implement strategies in Algerian hospitals to control the emergence of those microorganisms, including phenotypic and molecular tests to detect these bacteria.We emphasize that some measures must be taken to slow down the rising problem of such bacteria and to prevent nosocomial outbreaks which could lead to an endemic situation.

Table 1 .
Distribution of isolated Gram-negative bacilli (GNB) strains by species, wards, and type of sampling.

Table 4 .
Phenotypic and genotypic features of carbapenem-resistant Gram-negative bacilli clinical isolates.