Frequency of Multi-Drug Resistant Enterobacter Species Isolated from Patients with Different Clinical Manifestations in Khartoum State, Sudan

ABSTRACT


INTRODUCTION
The genus Enterobacter includes facultative anaerobic Gram-negative bacilli that are 2 mm long, motile by peritrichous flagella and belong to the family Enterobacteriaceae.Several strains of these bacteria are pathogenic and cause opportunistic infections in immunocompromised (Adeolu et al., 2016).It was first described in 1960, but changes in taxonomy have occurred in the last 50 years (Anne P et al., 2019).For example, E. sakazakii has been reassigned to a new genus Cronobacter (Anne P et al., 2019).
Enterobacter spp are natural commensals of the animal and human gut microbiota.Among these bacteria, only certain subspecies/species have been associated with hospital-acquired infections and outbreaks (Akbari M et al., 2015).
Enterobacter infections can include bacteremia, lower respiratory tract infections, skin and soft-tissue infections, urinary tract infections, endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis, central nervous system infections, and ophthalmic infections (Mezzatesta et al., 2012).Urinary and respiratory tracts are the most common sites of infection.Enterobacter infections do not produce a unique enough clinical presentation to differentiate them clinically from other acute bacterial infections (Mezzatesta et al., 2012).
Enterobacter species are members of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) which are described as the leading cause of resistant nosocomial infections (Akbari M et al., 2015), (Wu W, Feng Y, Zong Z. 2018).Enterobacter aerogenes, E. cloacae and E. hormaechei represent the most frequently isolated species described in clinical infections, especially in immunocompromised patients and those hospitalized in an ICU (Intensive Care Unit), due to the adaptation of these species to antimicrobial agents and their behavior as opportunistic pathogens.Several hospital outbreaks have been reported in Europe since the mid-1990s, and the wide use of extensive broad-spectrum antibiotics has stimulated the spread of resistant clones (Anne P et al., 2019).These pathogens are frequently associated with a multidrug-resistance phenotype, mainly due to their adaptation to the hospital environment and the pathogens' ability to easily acquire numerous genetic mobile elements containing resistance and virulence genes, which make their treatment difficult (Davin-Regli A, Pagès JM. 2015), (Davin -Regli A, Masi M, et al., 2016).Antibiotic resistance, regulation of resistant genes and the clinical implications of these situations have been extensively studied (Anne P et al., 2019).
The accurate identification of species and subspecies remains a challenge.The development of genome sequencing has rapidly modified the phylogeny of the genus, particularly that of the E. cloacae complex (Izdebski R et al., 2015).
Pathogenicity/virulence of this bacterium remains rather unclear due to the limited number of works performed to date in this field.In contrast, its resistance against antibacterial agents has been extensively studied.In the face of antibiotic treatment, it is able to manage different mechanisms of resistance via various local and global regulator genes and the modulation of the expression of different proteins, including enzymes (ß-lactamases, etc.) or membrane transporters, such as porins and efflux pumps (Anne P et al., 2019).

MATERIALS AND METHODS Study Design, Duration, and Population:
A cross-sectional laboratory-based study was conducted from February 2021 to October 2021.This study was conducted at Yastabshiroon Hospital and Ribat Teaching Hospital in Khartoum State, Sudan.The specimen was collected from Sudanese patients who were expected to have bacterial infections and had been examined by a physician.A total of three hundred and eighty-four (n=384) different clinical specimens (urine, wound swab, sputum, and blood) were collected in sterile containers.

Sampling Technique:
This study is based on the noprobability convenience sampling technique.

Ethical Clearance:
The study proposal was approved by the ethical board of Sudan University of Science and Technology and Research Committee, Ministry of Health.Informed consent was taken from each patient before enrollment into the study.

Isolation and Identification Schemes:
MacConkey agar media and Chromogenic agar media (Liofilchem Co. Italy) were used for the isolation of Enterobacter isolates.The medium was prepared and the plate was inoculated from urine, wound swabs, sputum specimens, and blood culture media, then the plates were incubated overnight at 37 0C.The isolate was identified according to lactose fermentation on MacConkey agar and the color produced on Chromogenic media.

Susceptibility test Enterobacter Species:
The susceptibility of isolates was determined by the agar diffusion method, using a modification of the Kirby-Bauer disc diffusion technique according to CLSI guidelines (CLSI, 2018).The following antibiotic discs (drug concentrations in μg) were used: Amikacin (30), Amoxicillin (30), Amoxyclav (30), Ciprofloxacin (5), Ceftriaxone (30), Cephotaxime (30), Ceftazidime (30), Cefotaxime (30), Cefepime (30), Chloramphnicol (30), Co-trimoxazole (30), Nitrofuratoin (30), Vancomycin (30), Tetracycline (30), Gentamicin (30), Imipenem (30), and Colistin.Enterobacterlike isolates from an overnight culture were used for the sensitivity test.A colony of the isolate was picked with a straight sterile wire, inoculated into sterile 5 ml peptone water, and shaken to dissolve it.The turbidity of the suspension was matched against 0.5 McFarland standards.Poured on a sterile Mueller-Hinton agar plate.The inoculated agar plate was swayed gently to ensure that the whole agar surface was covered.The plate was drained to remove excess fluid.The antibiotic discs were placed on the agar surface, leaving a space of 25mm between them, and pressed slightly to ensure sufficient contact with the agar surface.The plates were then incubated at 37 o C for 24 h.The plates were incubated inverted in the incubator at 37°C overnight.The plates were examined for inhibition zone size.Positive control (Enterobacter ATCC25922) and negative control (E. coli ATCC25922) were used to confirm the results.

Extended-spectrum β-lactamases (ESBLs) Confirmatory Test:
The presence of ESBLs was confirmed by Phenotypic Confirmatory Test method (PCT).The CLSI-ESBL phenotypic confirmatory test with Ceftazidime 30 μg, Cefotaxime 30 μg and Cefepime 30 μg was performed for all positive isolates by disc diffusion method on Mueller-Hinton agar plates with and without 10 μg of Clavulanic acid.A tube containing about 2 ml of sterile normal saline is inoculated with a pure culture growth until matching has occurred with 0.5 McFarland turbidity standard of approximately 1-2×108 CFU/ml.For each isolate one or more of the following cephalosporins disc ceftazidime (CAZ) 30 μg, cefotaxime (CTX) 30 μg and cefepime (FEP) was added to Mueller-Hinton agar plate seeded with bacterial suspension.On the same plate one or more of the antibiotics discs containing ceftazidime 30 μg+10 μg of clavulanic acid (CAL 30/10 μg), cefotaxime 30 μg+10 μg of clavulanic acid (CTL 30/10 μg) and cefepime 30 μg+10 μg of clavulanic acid (supplied by Liofilchem Co. Italy), were applied aseptically on the plate and pressed gently to agar surface using a sterile forceps.Within 15 min the plates were incubated at 37 °C for 16-18 hrs.A difference of ≥ 5 mm between the zone diameters of either of the cephalosporin disk and their respective cephalosporin/clavulanate disk is taken to be phenotypic confirmation of ESBLs production.

Modified Hodge Test:
The presence of carbapenemase enzyme was confirmed by Modified Hodge Test (MHT).A 1:10 dilution of inoculums of the indicator organism E. coli ATCC 25922, adjusted to a 0.5 McFarland turbidity standard, was used to inoculate the surfaces of plates containing Mueller-Hinton agar by swabbing.After the plates were allowed to stand at room temperature for 10 min, a disc containing meropenem (10 μg) were placed on the agar plates.Subsequently, three to five colonies of the test organisms (from an agar plate grown overnight) were inoculated onto the plate in a straight line out from the edge of the disk, using a loop.Quality control of the following organisms MHT Positive Klebsiella pneumoniae ATCC1705 and MHT Negative Klebsiella pneumoniae ATCC1706 were run with each batch of the test.Plates were examined after overnight incubation at 37°C.After 24 hrs, MHT Positive test showed a clover leaf-like indentation of the Escherichia coli 25922 growing along the test organism growth streak within the disk diffusion zone.MHT Negative test showed no growth of Escherichia coli 25922 along the test organism growth streak within the disk diffusion.

Data Analysis:
Data were analyzed using the statistical package for social science software (SPSS v. 20)

Bacterial Isolates:
Identification of the isolates was carried out by using colony morphology on MacConkey agar media following incubation for 24 h at 37 0 C, colonies were circular (1-2 mm in diameter), white, Opaque and elevated with regular margin lactose fermented, and on Chromogenic agar media Produced green color.As shown in Figure 2.

Gram's Stain and Biochemical Tests:
Microscopic examination showed that these bacteria were Gram-negative rodshaped (0.562-3 mm) and occurred singly, as shown in Figure 3-A 3-B.Catalase activity was determined by observing bubble formation after dropping H2O2 on 24 hrs old biomass on nutrient agar.Oxidase activity was tested by using an oxidase reagent (bioMe´rieux).in this study showed that all Enterobacter-like isolates were motile, catalase positive, and oxidase negative, lactose and glucose fermented with gas and noH2s production on kiligler Iron ager.While 21(95.4%)isolates were Indol (negative), 16(72.7%)isolates were Citrate (positive), and 16 (72.7%)isolates were Urease (negative).
In this study, we have shown a high prevalence of ESBLs and carbapenemase enzymes produced by Enterobacter spp in Khartoum.

Demographic Data of Patients with Enterobacter Spp Infections:
Among the 384 different clinical specimens, 22 isolates identified as Enterobacter spp, 11 (50%) were males while 11 (50%) were females.There was no significant difference in the distribution of isolates among the gender (p = 0.645).
The ages of patients were classified into five categories: the highest frequency was shown in the age group (15-30 years old, 46-60 years old, and age group >60 years old 6 (27.3%) the lowest frequency was shown in the age group <15 years old 1(4.5%),There was no significant difference in the distribution of isolates among the age group (p = 0.246).as shown in Table2.

Table 2: shows the demographic data of a patient with Enterobacter infection, including age and gender
Table 3 shows the distribution of Enterobacter spp among patient status, the frequency of patients suffering in different clinics (outpatient) was 7(31.8%), while 12(54.5%)were admitted to hospital(inpatient), and 3(13.6%)ICU patient.There was no significant difference in the distribution of isolates among the patient status group (p = 0.259).
This study showed that the distribution of Enterobacter species isolates was 12(54.5%) in admitted hospital patients (inpatients) including intensive care unit patients 3(13.6%), while 7(31.8%) of outpatients accounted for the least frequent samples, There was no significant difference in the distribution of isolates among the patient status group (p = 0.259), these findings disagree with a previous study conducted in Kermanshah, Iran by (Zeinab Mohseni et al;2021) who recorded most cases of positive

Frequency of Enterobacter spp among other isolated bacteria
Enterobacter were isolated from ICUs(ICU, NICU, and PICU;35.6%) and the emergency ward (bone marrow and kidney;1.3%)and outpatients (9.2%)accounted for the least frequent samples.

Conclusion
Enterobacter spp has emerged as a clinically significant cause of a wide variety of bacterial infections.There is still a lack of comprehensive molecular and clinical epidemiological analysis.
In this study, Enterobacter spp is the second most common cause of nosocomial Gram-negative bacillary infections (after E. coli.).
The results of this study indicated the high prevalence of Enterobacter species resistant to the majority of assessed antibiotics.In addition, prevalence rates of ESBL and carbapenemases producing Enterobacter species and MDR strains were high In Khartoum-Sudan.
Finally, Enterobacter species was relatively high in Sudan, and it seems that carbapenems cannot be considered the best drugs of choice for the treatment of MDR and ESBL-producing Enterobacter species.

Fig 1 :
Fig 1: The distribution of specimens.

Fig. 2 :
Fig. 2: Colonial morphology of isolated bacteria on MacConkey agar and Chromogenic agar media.A. Lactose fermented colonies produced by Enterobacter spp on MacConkey agar media.B. Enterobacter spp produced green color colonies on Chromogenic agar media.

Fig. 3 :
Fig. 3: A: showing Gram-negative rod-shaped (0.562-3 mm) and occurred singly.B: 24 hr old culture of isolated bacteria on kiligler Iron ager test show lactose and glucose fermented with gas and no production of H2s, Citrate Utilization test (positive), Urea Hydrolysis Test (negative), and Indol test (negative).
. A p-value of <0.05 was considered significant for all statistical tests in the present study.
. Basic biochemical characterization of the 22 Enterobacter-like isolates was achieved using the Catalase test, Oxidase test, kiligler Iron ager test, Motility test, Citrate Utilization test, Urea hydrolysis test, and Indol test, as shown in Figure

Table 3 :
Distribution of Patient status.

Table 4 :
Frequency of Enterobacter spp infections among significant growth.