Frequency of Isolation of Enterobacter Species from a Variety of Clinical Specimens in a Teaching Hospital in Nigeria

Purpose: To determine the frequency of occurrence of Enterobacter species and their antibiogram from clinical specimens of blood, cerebrospinal fluid, urine and wound obtained from University of Benin Teaching Hospital, Benin City, Nigeria. Methods: Specimens were obtained from patients who were seen at the various units of the hospital during the period January 2008 to June 2010. The total number of specimens was 6632, and were obtained from 1678 adult males, 2010 adult females and 2944 children. The specimens were collected prior to commencement of antibiotic therapy, and cultured immediately using standard bacteriological methods. Growths were identified by colonial morphology and characteristics, and biochemical reactions. Antimicrobial sensitivity test was performed according to Kirby-Bauer disc diffusion method as per Clinical and Laboratory Standards Institute (CLSI) recommendation. The control organism was a sensitive strain of Eschrichia coli (NCTC 10418) R esults: Two species of Enterobacter, namely, E. aerogenes (104; 97.2 %) and E. sakazakii (3; 2.8 %) were isolated from the four types of clinical specimens, accounting for 1.6 % of all the samples. Sensitivity to antibacterials was as follows: ceftazidime (55.0 %), ofloxacin (53.3 %) and amoxicillin clavulanate (48.3 %). They were strongly resistant to the other antibiotics used in the study, especially the cephalosporins. There was no significant difference in infection rate among the age groups (p > 0.05). However, there was significant difference (p < 0.05) between isolates from cerebrospinal fluid, on the one hand, and those from wound, urine and blood, on the other hand. Conclusion: The rate of isolation of Enterobacter species in the health facility was low. Remarkable drug resistance of the organisms make them clinically significant pathogens.


INTRODUCTION
Enterobacter aerogenes, E. Cloacae and E. sakazakii are commonly encountered Enterobacter spp. in most clinical specimens. These three species are differentiated by urease test and pigment production. E. cloacae is urease positive while the other two are negative. E. sakazakii produces yellow pigment which differentiates it from the other two species [1].
These organisms are Gram-negative capsulated bacilli and in the Petri dish, form round mucoid colonies; optimum growth temperature is 37 0 C . Enterobacter species are similar to Klebsiella species with large round mucoid colonies, but can be differentiated by a few tests such as motility and urease tests [2]. They are facultatively anaerobic, catalase positive, citrate positive, indole negative and oxidase negative. They ferment glucose and lactose with the production of acid, and are sucrose positive [2].
The organisms are distributed in water, soil, sewage, dairy products and vegetables, are part of the commensal enteric flora and usually are not pathogenic. However, some strains are known to produce shig-like toxin [3]. Enterobacter species have also been associated with nosocomial infections and a variety of opportunistic infections involving the urinary and respiratory tracts, and cutaneous wounds. E. sakazakii which is a pigmented strain of E. cloacae has been encountered in several cases of meningitis, bacteremia and sepsis [4]. It has also been associated with outbreak of necrotizing enterocolitis associated with the strain in powdered milk formula, and fatality rate is as high as 75 %. [5,6]. These enterobacter organisms cause significant morbidity and mortality. They can also cause community acquired infections resulting in endocarditis, intra-abdominal infections, septic arthritis, osteomyelitis and ophthalmic infections [7]. Risk factors for nosocomial infections include hospitalization for more than 2 weeks, invasive procedures and presence of central venous catheter, hands of hospital personnel and hospital equipments. Enterobacter species has an outer membrane that contains among other things lipopolysaccharides from which Lipid-A plays a major role in sepsis. Lipid-A, which is an endotoxin, is the major stimulus for the release of cytokines which are the mediators of systemic inflammation and its complications [8] Enterobacter species are resistant to most antibiotics, especially the cephalosporins. Their resistance to β-lactam antibiotics, chloramphenicol, quinolones and tetracyclines have been well documented [9]. Reports abound in the literature on the increasing resistance of Enterobacter species to the penicillins and all generations of cephalosporins, and their emergence in clinical specimens.
The paucity of information on the clinical significance of the Enterobacter species in Edo State south-central Nigeria, necessitated this study, the aim of which was to determine the frequency of isolation of Enterobacter species and their antibiogram from clinical specimens obtained from University of Benin Teaching Hospital, Benin City, Edo State, Nigeria.

Study setting
The study, which was prospective and crosssectional, was carried out at the University of Benin Teaching Hospital (UBTH), Benin City, Nigeria. The hospital has a bed complement of six hundred (600) and an equipped microbiology laboratory that provides laboratory services to the neighbouring states of Ondo, Delta, Kogi and Anambra.

Sampling method
The samples used in the study were obtained consecutively from patients who were seen at the various facilities in the hospital during the

Wounds
Wound swab was cultured on blood and MacConkey agars. The specimens were aseptically inoculated on the media and streaked to obtain distinct colonies [10]. After inoculating on media, a grease-free slide was smeared for Gram staining and microscopy. Two blood agar plates inoculated with specimen were incubated -one aerobically and the other anaerobically. The anaerobic incubation was done using a gas pact in an anaerobic jar. The pact was cut on one side to introduce 10 ml of water and placed in a jar along with the plates for anaerobic incubation. The jar was closed and was not opened until 24 h later.

Blood
Blood samples were aseptically collected from patients, 1 ml of which was aseptically introduced into nutrient and thioglycollate broths in bottles and incubated at body temperature (37 o C). The bottles were examined daily for growth (which manifests as turbidity or gas bubbles). Where growth was detected, the nutrient broth was subcultured on chocolate, blood and MacConkey agars and incubated aerobically while thioglycollate broth was subcultured on blood agar and incubated anaerobically as described above. Following subculture, Gram staining and microscopy were carried out.

Cerebrospinal fluid
Cerebrospinal fluid (0.5 ml) was obtained through a spinal tap and cultured immediately on chocolate, blood and MacConkey agar plates. Gram staining and microscopy were carried out. The culture on chocolate plate was incubated in 5 -10% carbon dioxide environment for 24 h at 37 o C.

Urine
Urine was obtained in a 20 ml sterilin container and after thorough mixing, a wire loop (30 morse gauge) with a chamber of 3.26 mm circumference was used to streak the mixed urine on blood, MacConkey andCLED agars, and incubated aerobically for 24 h. All the plates were incubated at a temperature of 37 o C. Isolates were identified based on colonial morphology, motility, Gram staining, oxidase and biochemical reactions using the protocol of Cowan and Steel [2,11].
Isolates from 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, shaken to dissolve it and poured on a sterile nutrient 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 inhibition zone diameter (IZD) was measured, and IZD < 5 mm was taken as resistance while IZD > 5 mm was considered sensitive.

Statistical analysis
Gen Stat software (release 8.1) was used to analyze the data obtained. Differences in the distribution of isolates among the age groups as well as among the clinical specimen types were determined while statistically significant differences were computed Student's t-test and analysis of variance (ANOVA). Differences between specimen types (p <0.05) was determined using Duncan multiple range test.

RESULTS
The specimens (6632) yielded 107 (1.6%) isolates of enterobacter species.There were 104 Enterobacter aerogenes and 3 Enterobacter sakazakii spp. The distribution of organisms among the age groups are shown in Table 1. There was no significant difference in the distribution of isolates among the age groups (p < 0.05). However, as Table 2 shows, there was a significant difference between isolates from cerebrospinal fluid and those from other specimens (p < 0.05) except for the isolates from blood specimens.
The susceptibility of the Enterobacter isolates to various antibiotics is shown in Tables 4  and 5.
Based on the antibiogram, Enterobacter aerogenes was moderately sensitive to ceftazidime, ofloxacin and amoxicillin clavulanate while showing strong resistance to the other drugs. Enterobacter sakazakii, on the other hand, exhibited higher susceptibility to the antibiotics except erythromycin, cloxacillin and oxacillin. Enterobacter aerogenes, which was isolated from urine, demonstrated good susceptibility to nitrofuradantoin but was moderately susceptible to ceftazidime and ofloxacin.       [13]. E aerogenes, which was the predominant isolate in our study, is commonly encountered in clinical specimens. It is known to be associated with a variety of opportunistic infections [3]. It is associated with infections involving the urinary and respiratrory tracts, and cutaneous wounds, and occasionally causes sepsis and meningitis. E. sakazakii which was scantily encountered in the study has been reported in several cases of neonatal meningitis and sepsis, and has a high fatality rate [4]. Thus, the organism can be highly virulent [5,6]. Recent reports have linked E. sakazakii infection in neonates with contaminated powdered milk formulas [14].
The epidemiology of Enterobacter infection as seen from this study appear to place children at relatively higher risk of infection, followed by adult females, with adult males least predisposed to infection. However, the differences were not statistically significant (p > 0.05). This mean that all age groups and gender types are equally predisposed to infection. Children are, however have been known to be most susceptible to any infection as a result of habits, exposure and lower immunity. Enterobacter infection is largely associated with decreased immunity. Those heavily affected are the immunocompromised as well as factors such as increased length of stay in the hospital; increased length of stay in the intensive unit; those on urinary catheter and long term antibiotic administration [15]. The distribution of enterobacter species in the clinical specimens indicated a significant difference among the specimen types. There was statistically no difference in the distribution of isolates in the other specimen categories. Thus cerebrospinal fluid (CSF) is the least likely to be infected Enterbacter spp. This may be due to the fact that organisms do not easily gain access to the cerebrospinal fluid as a result of some sort of barrier.
The antibiogram showed that Enterobacter aerogenes exhibited high resistance to the antibiotics used in the study; these are also the antibiotics commonly used in these setting for the treatment of infections. The observed resistance is agrees with literature reports which claimed that Enterobacter species are reisistant to most antibiotics. The resistance of this species to β-lactam antibiotics, chloranphenicol, quinolones and tetracycline is well documented in scientific literature [9]. There are numerous reports in the literature on the increasing resistance of Enterobacter species to penicillins and all generations of cephalosporins and their emergence in clinical specimens [9].
The antibiogram of Enterobacter sakazakii showed the organism to be more susceptible to most antibiotics, except oxacillin and cloxacillin Enterobacter aerogenes was the only species of Enterobacter that was isolated from urine specimens and they exhibited a slightly different antibiogram. They were less susceptible than those from wound, blood and cerebrospinal fluid. However, the high susceptibility to furadantoin is an indication that the drug could be the drug of choice for any Enterobacter urinary tract infection. They were moderately susceptible to ceftaxidime and ofloxacin. The high resistance to other drugs in the antibiogram should be a cause for concern.

Pathology issues
The Enterobacter species, like other members of Enterobacteriaceae family contain an outer membrane which contains, among others, lipopolysaccharides of which lipid-A plays a major role in sepsis. Lipid-A, which is an endotoxin, is the major stimulus for the release of cytokines which are the mediators of systemic inflammation and its complications [8]. In the present study, ceftaxidime (a cephalosporin) and ofloxacin (a quinolone) exhibited some moderate efficacy and thus could be effective in the management of Enterobacter infection. This observation contradicts some literature claims which reported that Enterobacter spp are resistant to these drugs [9].
The high resistance of Enterobacter organisms is associated with the production of extended spectrum β-lactamase which confers on them resistance to many antibiotics. β-Lactamases are the major defense systems of Gram-negative bacteria [16]. Since β-lactam antibiotics came into clinical use, β-lactamase evolved with them [17]. It is very likely that Enterobacter species acquired their resistance to drugs through the production of this enzyme, β-lactamase. Worst still, enterobacter species together with some members of Enterobacteriaceae carry a gene for chromosomally encoded βlactamase. Unlike the plasmid-mediated gene, the enzyme is not usually expressed in the uninducible form. They become induced by antibiotics, body fluids and some amino acids. It is a concern that organisms such as Enterobacter aerogenes harboring genes for inducible β-lactamase may show false susceptibility if tested in the uninduced state [16]. This may explain the causes of some treatment failures as the organism develops resistance during therapy. For this reason, management of any enterobacter infection should be done with caution.
Risk factors for enterobacter infections include not only long hospitalization and presence of indwelling catheters since infections have also been traced to equipment, contaminated objects and hands of hospital personnel. The risk factors for extended spectrum beta lactamase (ESBL)producing organisms are little different from the risk factors of other nosocomial infections [18]. In outbreak situations, successful intervention has usually involved the institution of barrier precautions in the form of hand washing, as well wearing of cloves and gowns. Presently, there are no β-lactam antibiotics in development that can treat infections caused by organisms producing some of the new β-lactamases. The available agents should be used judiciously and effective control measures should be implemented in situations of outbreak to prevent further spread of the pathogens.

CONCLUSION
The frequency of isolation of Enterobacter species from clinical specimens obtained from University of Benin Teaching Hospital, Nigeria was low. Virulence and remarkable drug resistance of the organisms make them clinically significant pathogens.