Antibiotic susceptibility patterns of pathogens isolated from laboratory specimens at Livingstone Central Hospital in Zambia

Background Multidrug resistance (MDR) is a global problem that require multifaceted effort to curb it. This study aimed to evaluate the antibiotic susceptibility patterns of routinely isolated bacteria at Livingstone Central Hospital (LCH). Methods A retrospective study was performed on all isolated organisms from patient specimens that were processed from January 2019 to December 2021. Specimens were cultured on standard media and Kirby-Bauer disc diffusion method was employed for susceptibility testing following the Clinical and Laboratory Standard Institute’s recommendations. Results A total of 765 specimens were processed and only 500 (65.4%) met the inclusion criteria. Of the 500, 291(58.2%) specimens were received from female and from the age-group 17–39 years (253, 50.6%) and 40–80 years (145, 29%) in form of blood (331, 66.2%), urine (165, 33%) and sputum (4, 0.8%). Amongst the bacterial isolates, Staphylococcus aureus (142, 28.4%) was the commonest followed by Escherichia coli (91, 18.2%), and Enterobacter agglomerans (76, 15.2%), and Klebsiella pneumoniae (43, 8.6%). The resistance pattern revealed ampicillin (93%) as the least effective drug followed by oxacillin (88%), penicillin (85.6%), co-trimoxazole (81.5%), erythromycin (71.9%), nalidixic acid (68%), and ceftazidime (60%) whereas the most effective antibiotics were imipenem (14.5%), and piperacillin/tazobactam (16.7%). The screening of methicillin resistant Staphylococcus aureus (MRSA) with cefoxitin showed 23.7% (9/38) resistance. Conclusion Increased levels of MDR strains and rising numbers of MRSA strains were detected. Therefore, re-establishing of the empiric therapy is needed for proper patient management, studies to determine the levels of extended spectrum beta lactamase- and carbapenemase-producing bacteria are warranted.


Introduction
Antibiotics are essential commodities in managing bacterial infections in humans and animals. However, the spread of antibiotic-resistant bacteria had been increased by the overuse and misuse of antibiotics as well as social and economic factors [1]. Therefore, antibiotic resistance is a public health concern that has been recognized globally. Studies have indicated that a lot of lives are lost due to antimicrobial resistance related illnesses every year, and almost 100% β-lactams and some 3rd generation cephalosporins and carbapenems have recorded resistance by some organisms [2].
Prior to the development of the first β-lactam (penicillin) and its release in medical practice, emergence of resistance had been reported and the first β-lactamase enzyme was identified in Escherichia coli(E.coli) [3]. Extended Spectrum β-lactamases (ESBLs) are enzymes that deactivate a variety of β-lactam antibiotics including penicillins, cephalosporins (3rd and 4th generations) and monobactams but less likely to deactivate cephamycins like cefoxitin [3][4][5]. Furthermore, the production of carbapenemase enzymes by some Enterobacteriaceae is alarming as these enzymes can break down antibiotics including carbapenem antibiotics, which are typically reserved to treat multidrug-resistant bacterial infections [6]. Carbapenem-resistant Enterobacteriaceae (CRE) causes very hard to treat infections because of being resistant to carbapenems that are used on multidrug resistant strains [7]. It is sufficed to note that the more bacteria get exposed to antimicrobial agents the more resistance develops. Therefore, there is need to preserve the current antimicrobial agents by using them judiciously otherwise, we risk having pandemic AMR infections that will devastate the global community.
In Africa, especially in low and medium-income countries (LMIC), antimicrobial resistance monitoring is inadequate, but the extensive usage of antibiotics to prevent and treat infectious diseases has led to the emergence and spread of antibiotic resistance which has influenced a particular force on susceptible bacteria leading to resistant strain survival, consequently increasing medical costs, illnesses and deaths of patients [8]. Therefore, antibiogram studies in LMIC are important to closely monitor the trends of AMR at hospital and national levels. This retrospective study aimed to assess antibiotics resistance among commonly isolated bacteria from routine specimens at Livingstone Central Hospital from 2019 to 2021.

Study design and site
A retrospective cohort study was conducted at Livingstone Central Hospital (LCH) on routine specimen isolates from paediatric and adults who visited the hospital between January 2019 -December 2021. Livingstone Central Hospital has different departments that include laboratory, internal medicine, surgery, paediatrics, and obstetrics and gynaecology. The LCH microbiology laboratory participates in a bacteriology External Quality Assessment (EQA) program and has been accredited by the Southern African Development Community Accreditation Service (SADCAS).

Eligibility criteria
This study included all specimens having information on the patient's gender, age, location (ward/clinic), name of organism and antibiotic susceptibility testing. However, any isolated organism without a species name, and with unknown source (i.e., lack of age-, location-, and gender of patient, and sample type) were excluded from the study.

Bacterial culture and Identification
The bacteriological analysis involves culturing of specimen on appropriate quality controlled culturing media following the national standard operating procedures, and Clinical and Laboratory Standards Institutes (CLSI) guidelines [9]. All routine specimens from inpatients and outpatients were cultured on MacConkey, blood, and chocolate agar plates and incubated at 37˚C in an appropriate condition. Where applicable selective media such as mannitol salt agar and xylose lysine deoxycholate agar were used. Identification of bacterial isolates was aided with a Gram staining method, culture characteristics, motility test, and various biochemical tests such as oxidase test, triple sugar iron (TSI), indole production test, urease production test and citrate utilization test as described previously [9][10][11].

Quality control (QC)
Escherichia coli ATCC 25922 and S. aureus ATCC 25923 were used as QC for all antibiotics and culture media.

Data collection and analysis
Data from electronic laboratory system generated reports on all isolated organisms at LCH microbiology laboratory for three years (January 2019 to December 2021) was used from which information such as age, gender, patient's location, name of the organism and the antibiotic susceptibility were considered. The collected data was entered, assorted, and coded using Microsoft Excel 2019 and then exported to Statistical Package for Social Science (SPSS) version 20 for analysis. Descriptive statistics was used to describe our data. Microsoft Excel 2019 and Graph prism 5 were used for graph generation. A chi-square test was used for categorical variables and a p-value of �0.05 was considered statistically significant.

Ethical consideration
This is a retrospective study that analysed secondary data generated from routine laboratory specimens from both adult and paediatric departments. There was no human interaction, and no personal identifiers were included in this study. However, ethical waiver was granted by Mulungushi University School of Medicine and Health Sciences Research Ethics Committee (SMHS-MU2-2021-33v1) while permission to use the Disa � Lab system generated data was obtained from Livingstone Central Hospital management.

The association of patient gender, age group, location, and year of isolation with resistant pattern of bacteria isolates
We conducted a chi-square test to determine the link between resistance pattern of bacteria and independent variables. We found that the potency of both nitrofurantoin and cefuroxime were affected by gender. Specifically, the effectiveness of nitrofurantoin on E. agglomerans (p = 0.041), co-trimoxazole on coagulase negative Staphylococci (CoNS, p = 0.044), cefuroxime on E. coli (p = 0.011) and cefotaxime on K. oxytoca (p = 0.026) as presented in Table 1. The patient location impacted negatively on the performance of chloramphenicol and ciprofloxacin against E. agglomerans (p = 0.002) and K. pneumoniae (p = 0.021), respectively (Table 1). Further analysis revealed that the E. aerogenes resistance to co-trimoxazole (p = 0.005) varied with year of isolation, and so was S. aureus resistance to tetracycline (p = 0.009), chloramphenicol (p = 0.002) and co-trimoxazole (p = 0.041). Lastly, S. aureus resistance to tetracycline was affected by specimen type (p = 0.047; Table 1).
However, S. aureus resistance to chloramphenicol had a significant reduction from 2019 to 2021 (p = 0.006) whereas its resistance to penicillin was the highest in 0-16years age group (p = 0.012, Table 2).

General characterization of bacterial isolates from routine specimens
Antimicrobial resistance (AMR) is a global public health problem that requires concerted effort from all sectors of life to control it. Due to lack of data on prevalence rates because of limited or absent antimicrobial surveillance systems and poor resources for antimicrobial susceptibility testing, AMR is a significant problem in sub-Saharan Africa [12]. Literature indicates that multidrug resistance is prevalent among gram-negative and gram-positive bacterial pathogens causing common infections irrespective of whether community-or hospitalacquired [12,13]. The One Health concept that focusses on consequences, responses, and actions at the animal-human-ecosystems interfaces, is needed to mitigate this issue and hence deserves full support from policymakers and stakeholders. Therefore, the current study aimed to evaluate the antibiotic resistant patterns of pathogens isolated from routine laboratory specimens from 2019 to 2021 at LCH as a means of understanding the AMR trends at the hospital. Generally, our study revealed that blood specimens were the most frequently processed specimen with 66.2% followed by urine (33%) and sputum (0.8%) indicating bacteraemia and urinary tract infections as the commonly managed infections at LCH. A similar study conducted in Ndola, Zambia found urine and blood specimens as the common processed specimens at Ndola Teaching Hospital [14]. Furthermore, many specimens came from out-patient department (64.7%), and we further noted that the influx of specimens to the laboratory reduced from 35% (for 2019 and 2020) to 30% (for 2021), conceivably because of the COVID19 pandemic that caused Government to put the country on periodic lockdown, thereby reducing the number of patients that accessed health services.
Several studies reveal that sepsis due to bacterial infections emanates from urinary tract infections which are more prevalent in women [19][20][21]. So, we expected to observe the similar trend in this study. Surprisingly, the potency of nitrofurantoin on E. agglomerans (p = 0.041), cotrimoxazole on CoNS (p = 0.044), cefotaxime on K. oxytoca (p = 0.026), and cefuroxime on E. coli (p = 0.011) were significantly reduced in male patients. This observation required further characterization of blood and urine isolates to clearly understand this phenomenon. Furthermore, the potency of chloramphenicol on E. agglomerans (p = 0.002) and ciprofloxacin on K. pneumoniae (p = 0.021) were significantly high among in-patients, which agreed with another study done in Kuwait [22]. Finally, cotrimoxazole potency on S. aureus isolates from blood specimens were significantly (p = 0.005) low. Therefore, the use of antibiotics especially from male admitted patients should always be proven by susceptibility testing. The resistant trend observed in this study clearly indicate possible prevalence of extended spectrum beta lactamases (ESBLs) [23], and E. agglomerans, K. pneumonia and S. aureus are clinically important pathogens that can develop multidrug resistance and cause difficult to treat infections in all age groups [24,25]. Imipenem is a carbapenem that shows broad-spectrum activity against gram-positive, gram-negative, and anaerobic bacteria, and is active against cephalosporinresistant Enterobacteriaceae producing ESBLs, whereas piperacillin/tazobactam is the combination of a fourth generation, extended-spectrum penicillin and a beta-lactamase inhibitor that is also effective against β-lactamase producing penicillin-resistant bacterial species [26,27]. Due to the wide spread of antibiotic resistance resulting from ESBL producing Enterobacteriaceae, carbapenems should normally be reserved as alternative treatment for such infections [28] but our study found that both imipenem and piperacillin/tazobactam were broadly used in almost all infections. Despite imipenem being observed as the most effective antibiotics at this LCH, there could be chances of irrational use of the antibiotics subsequently posing a risk of developing carbapenem-resistant bacterial strains. Also, the resistant pattern of isolated strains suggests prevalence of ESBL-producing Enterobacteriaceae that warrant further investigations. To circumvent the issues of carbapenem resistant Enterobacteriaceae and ESBL-producing Enterobacteriaceae, antimicrobial stewardship program should be implemented to curb purported irrational use of antibiotics and enhance adherence to proposed guidelines on antimicrobial use [29]. Additionally, the need for more studies (especially molecular studies) to further assess the prevalence of carbapenemases and redesign the empiric therapy to safeguard few potent carbapenems cannot be over emphasized.

Characterization of bacterial pathogens from paediatric department
Antimicrobial resistance impacts all population and the increasing trend of drug resistant infections in infants and children is usually unrecognized while some studies have shown prevalence of ESBL producing organisms [30,31]. Because of this scarcity of AMR data in children, we decided the characterize bacteria isolates from paediatric department specimens.

Characterization of bacterial isolates from in-patient department
Hospital acquired infections (HAIs) are of major safety concern for both health care providers and the patients [33]. These infections are usually acquired after hospitalization and manifest 48-72 hours after admission to the hospital. Even though it was difficult to determine whether the infections were hospital-acquired or not, due to the retrospective nature of our study, we thought of having a glimpse on the resistant levels of bacteria isolated from IPD. We noticed that more blood and urine specimens came from female patients. The most isolated bacterium was S. aureus (28.2%), followed by E. coli (18.1%), E. agglomerans (14.7%), K. pneumoniae (6.8%), CoNS (4.5%), and K. oxytoca (4.5%). The study revealed that ampicillin (93.4%) was the least effective drug followed by penicillin (83%), co-trimoxazole (82.5%), oxacillin (80%), erythromycin (74.4%), nalidixic acid (70.7%), cefuroxime (60%), and tetracycline (48.5%) whereas the most effective antibiotics were norfloxacin (18.2%), imipenem (18.5%) and clindamycin (28.6%). The picture of multidrug resistant strains was observed amongst IPD specimens and this calls for an in-depth analysis of antibiotic susceptibility patterns of nosocomial pathogens. The prevalence of gram-negative HAI-and carbapenem resistant-pathogens have been reported across the globe [34,35] and the cases are steadily increasing, posing a risk on the cost and effective management of HAIs. Therefore, antimicrobial stewardship programs [29,33] should be implemented in referral hospitals in Zambia and beyond.
In conclusion, our study has revealed the proportion of antibiotic resistance of commonly isolated bacteria at LCH and the importance of periodic monitoring of antibiotic resistance patterns in hospitals. Antibiogram studies can aid in selecting an effective antibiotic therapy, tracking local antibiotic resistance trends, and identifying antimicrobial stewardship initiatives and targets for education and improvement. The study discovered the presence of MDR strains among adult and paediatric in-patients and out-patients, an indication that the choice of antibiotic therapy at LCH may be reducing. Therefore, more studies to establish the prevalence of ESBL-and carbapenemase-producing bacteria for empiric therapy redesigning are warranted as monitoring data on antimicrobial susceptibility of common bacterial organisms is crucial for decision-making and quick detection of AMR at hospital levels. Moreover, identifying specific hospital wards and clinics with huge antibiotic resistance burden will be helpful in strategizing on an effective infection prevention method, as it was not established in our study.