This study was a cross-sectional study of all the patients at both male and female adults in the medical emergency unit of Alex-Ekwueme Federal University Teaching Hospital Abakaliki, Ebonyi State. The clinical diagnosis of infection was made by the emergency physician and casualty officers working in emergency unit. Samples were collected from patients based on the diagnosis and site of infection before administration of antibiotics. Clinically relevant specimens were collected from the participants that consented to be part of the study. The specimens were analyzed according to the microbiological standard protocol. Multidrug resistant phenotypes were determined using double disc synergy test and modified carbapenemase inactivation method (MCIM) for extended spectrum beta-lactamase and carbapenem resistance respectively. The data collected were analyzed using SPSS version 25.

This study was conducted at the medical emergency units of the Alex-Ekwueme Federal University Teaching Hospital Abakaliki (AEFUTHA), Ebonyi State. The hospital is the largest tertiary hospital in the Southeast of Nigeria. Ebonyi State is one the states in Southeast Nigeria, has Enugu and Benue towards north and southern border with Imo, Abia and Cross River.

Clinically relevant samples including blood, urine, wound swab, wound biopsy, sputum was collected based on the nature and sites of infection in order to increase the chances of isolating the pathogen of interest.

Adults on admission in emergency unit were recruited for this study after informed consent. Demographic data and clinical information of patients was obtained from the patient’ medical record file.

All eligible patients in admitted in emergency unit were included in the global point prevalence survey. While the sample size for the cross-sectional study was calculated based on the formula:

N = the desired sample size;

Z = the standard normal deviation, usually set at 1.96 corresponding to 95% Confidence interval;

p = the prevalence;

q = 1 − p;

d = the standard error (margin of error) set at 0.05.

Where prevalence rate of Klebsiella species infection is 16% in study done by Mike-Ogburia et al. (2025) [6]. The sample size was calculated using the formula above where: z = 1.96; p = 0.16; q = 1 − p = 0.84.; d = 0.05.

N = (1.96)² × 0.16 × 0.84/(0.05)²

= 3.8416 × 0.1344 0.0025

= 207 isolates

All patients with clinical diagnoses of infections were enrolled into the study.

Patients without clinical evidence of infection and those who were critically ill were excluded from the study.

A structured questionnaire was administered to patients to obtain demographic and medical record review which captured data on age, sex and clinical symptoms and length of hospitalization of the patients.

Blood culture bottle was given to the attending physicians to collect blood sample from adults on admission following examination by the attending physicians. Sterile bottle was given to patients with urinary tract infections for urine sample collection. Wound swabs were collected from patients with any form of skin and soft tissues infections, pus, cerebrospinal fluid and all samples collected were transported to the Microbiology laboratory of the Alex-Ekwueme Federal University Teaching Hospital Abakaliki Research Laboratory.

Quality control strains were obtained from Department of Medical Microbiology, AEFUTHA. Quality control of Antimicrobial Susceptibility Testing was carried out according to the clinical Laboratory Standards Institute (CLSI) guidelines using ATCC 43300 S.aureus as control strain [17].

Consecutive Sampling technique was used to select patients that met the eligibility criteria, subsequently patients with identified Klebsiella pathogens were recruited into the study. Non duplicate Klebsiella species isolates were used in the study.

Sputum, Wound Biopsy, Urine, Wound Swab Specimen Processing

Specimens were inoculated on blood agar (incubated for 24 hours) and MacConkey agar (incubated under 37˚C for 24 hours).

2.12.1. Blood Culture Processing

Incubated the blood culture bottles in the BACT ALERT machine at 37˚C, subcultured broth from a positive flagged on chocolate (incubated under 5% carbondioxide for 24 hours) and macconkey agar (incubated under 37˚C for 24 hours).

2.12.2. Gram Stain and Biochemical Identification of the Isolates

The isolates were Gram stained and Gram negative bacilli were selected for further identification. Klebsiella species were biochemically identified using chromogenic sugar fermentation method.

Microbact 20A/E (Oxiod) identification profile was used in identification of Gram-negative organisms to species level according to the manufacturer’s instruction. Oxidase test was used to select the type of microbact kit used for each of the isolate. Microbact 20A/E was used for oxidase negative Gram negative bacilli (Klebsiella spp.).

Principle: microbat 20A/E has commercially prepared reagents and biochemical sugars embedded on the wells of the kit. The organisms either ferment, or oxidize the sugar which leads to colour change seen after 24 hours incubation.

Procedure: the isolate was used to make a broth suspension of equal turbidity with 0.5 McFarland standards. The suspension was also used to fill the micro wells in the microbact kit. This was incubated for 18 hours in an ambient air between 35˚C to 37˚C. The manufacturer’s instruction was followed strictly and color changes read with the corresponding guide.

Result: the organism was identified with the codes generated using the microbact software.

2.12.3. Antibiotic Susceptibility Testing

Antibiotic susceptibility testing was done using Modified Kirby-Bauer disc diffusion method.

Principle: modified Kirby-Bauer disc diffusion relies on diffusion of the antibiotics impregnated in the disc into the agar to inhibit the bacterial growth and it is measured under standard conditions.

Procedure: an overnight growth and pure culture was used to make a suspension of equivalent turbidity with 0.5 McFarland standards. Mueller Hinton agar was used. The suspension of the organism was inoculated on the agar using sterile swab. The number of antibiotics disc used were maximum six discs in a 100 mm agar plate. The agar was incubated at 35˚C to 37˚C for 18 hours in an ambient air and the zone diameter (zone of inhibition) was measured using ruler.

Result: the zone of inhibition was interpreted as sensitive, intermediate (the intermediate category implies clinical efficacy in the body sites where the drugs are physiologically concentrated and also include a buffer zone for inherent variability in test methods) or resistant according to Clinical Laboratory Standards Institute (CLSI) standards [17].

Choiceofantibiotics: antibiotics used for different organisms were chosen based on CLSI and the antibiotics commonly prescribed in medical ward. Two Mueller Hinton agar plates were used for each isolate and maximum of six antibiotic discs were placed in 100 mm agar plate. The selected antibiotics for each of the organisms according to CLSI were as follows:

The Gram negative isolates such as Klebsiella spp.: gentamicin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), cefuroxime (30 μg), cefotaxime (30 μg), ceftriaxone (30 μg), cefepime (30 μg), piperacillin-tazobactam (100/10μg), amoxicillin-clavulanate (20/10μg), and meropenem (10 μg).

Multidrug Organism (MDRO): can be defined as resistance to at least one in three different classes of antibiotics. There are specific of multidrug resistance organism with enzymes that confer them ability to resist most classes of antibiotics such as carbapenemase producing Enterobacteriaceae (CPE), extended spectrum beta-lactamase (ESBL) and others [18][19].

2.13.1. Antibiotic Resistance Testing

The common resistance phenotypes tested on Klebsiella species were as follow: extended spectrum β-lactamase producer and carbapenemase producing Klebsiella.

2.13.2. Phenotypic Detection and Confirmation of Extended Spectrum β-Lactamase (ESBL) Producing Klebsiella spp. Using CLSI Break Point and Double Disc Synergy Test

ESBLs are enzymes produced by some Klebsiella spp. which hydrolyze most penicillin and cephalosporin up to fourth-generation as well as aztreonam.

Principle: the confirmation test for ESBL producing Klebsiella example double disc synergy test evaluates the synergy between an oxyimino cephalosporin and an inhibitor like clavulanic acid. The clavulanic acid inhibits ESBL and allows the third generation cephalosporin to inhibit the growth of the organism in that direction therefore forming a dome bell shape.

Screening: the Klebsiella isolates were screened for ESBL production by using disc diffusion of ceftazidime (30 µg) and cefotaxime (30 µg) placed on inoculated plate containing Muller Hinton agar according to CLSI recommendation. The zone diameter of ≤22 mm and ≤27 mm for ceftazime and cefotaxime respectively were indicated as suspected ESBL production. The positive isolates were subjected to confirmatory test using double disk synergy test.

Procedureoftheconfirmatorytest: double disc synergy test (DDST) was used to test for presence of ESBL in Klebsiella. Discs containing cephalosporin (cefotaxime and ceftazidime) were placed next to a disc with clavulanic acid (amoxicillin-clavulanic acid). The distance between the discs was 20 mm centre to centre. The agar was incubated at 35˚C to 37˚C for 18 hours in an ambient air.

Result: positive result was indicated by augmenting of zone of inhibition towards the direction of the amoxicillin-clavulanic acid with dumb-bell or keyhole appearance.

QualityControl

Klebsiellapneumoniae ATCC 700603 was used as positive control organism.

Escherichiacoli ATCC 25922 was used as negative control organism [17].

2.13.3. Phenotypic Screening and Confirmation of Carbapenemase Production in Klebsiella spp.

Carbapenemases are β-lactamases that hydrolyze penicillin, in some cases cephalosporin, monobactams and carbapenems. The technique used to screen for carbapanemase production in enterobacteriaceae was the result of the disc diffusion test of the isolates on carbapenems (meropenem) and confirmation was done using Modified Carbapenem Inactivation Method (mCIM).

Principle: modified carbapenem inactivation method uses the principle of detection of enzymatic hydrolysis by incubating a carbapenem with a bacterial suspension. The principle involves incubation of carbapenem disc for four hours in an aqueous suspension of a carbapenemase-producing Klebsiella spp., the carbapenem in the disc is degraded by the carbapenemase; as against, if the test organism does not produce carbapenemase, the carbapenem retains its antimicrobial activity after incubation in the bacterial suspension. The disc is removed from the suspension and placed on a Mueller Hinton agar swabbed with indicator organism; following overnight incubation, the zone of inhibition is measured to determine if the carbapenem have been hydrolyzed.

Screening: the Klebsiella isolates were screened for carbapenemase production by using disc diffusion of meropenem (10 µg) placed on inoculated plate containing Muller Hinton agar and the isolate according to CLSI recommendation. The isolates that were either resistant or intermediate for meropenem or that is with zone diameter ≤ 23 mm (equivalent of 2 - 4 µg/l MIC) for meropenem were subjected to confirmatory test using modified carbapenem inactivation method.

Procedureoftheconfirmatorytest: the isolate to be tested was emulsified in 2 ml tryptic soy broth (TSB) and vortex for 15 seconds. The carbapenem (meropenem) disc was added to the TSB tube using sterile forcep and incubated for four hours at 35˚C to 37˚C in ambient air. The standardized inoculum of the indicator organism (E. coliATCC29522) was used to inoculate on Mueller Hinton agar. The meropenem was removed from the TSB using 10 μL loop by placing the flat side of the loop against the flat edge of the disc and carefully placed on the inoculated Mueller Hinton agar (MHA). The MHA plate was incubated at 35˚C to 37˚C in ambient air for 18 - 24 hours. Following incubation, the zone of inhibition was measured.

Result: carbapenemase production was indicated when the zone diameter is 6 - 15 mm or presence of colonies within a 16 - 18 mm zone and was reported as carbapenemase detected. It is indicated as negative, if the zone is ≥19 mm and was reported as carbapenemase not detected.

QualityControl:K.pneumoniae ATCC BAA-1705 was used as positive control, K.pneumoniae ATCC BAA-1706 as negative control and E.coli ATCC 25922 served as indicator organism [17].

The statistical analysis software version 25 (SPSS) was used for data analysis.

The data were entered into Excel Microsoft and statistical analysis was done using Social Sciences (SPSS) software, version 25. The variables were expressed in percentages and frequency tables.

The ethical approval for the study conduction was obtained from Alex-Ekwueme Federal University Teaching Hospital Abakaliki Research Ethical Committee (HREC). The study was conducted according to declaration of Helsinki. Both verbal and written informed consents were obtained from all the participants after the detail of the study was explained to them.