Extent and Resistance Patterns of ESKAPE Pathogens Isolated in Pus Swabs from Hospitalized Patients

Antimicrobial resistance has persisted as a global threat with increasing associated numbers of morbidity and mortality. ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) were termed by the Infectious Diseases Society of America as a group of bacteria with rapid antibiotic resistance development. The aim of the study was to describe the extent and resistance patterns of ESKAPE pathogens isolated in pus swabs from patients admitted at Muhimbili National Hospital, Tanzania. A retrospective cross-sectional study was performed in August 2019. A total of 75 admitted patients with open wounds and surgical site infections were recruited. Files were analyzed to collect microbiology laboratory data and relevant patient data. A total of 76 clinically significant bacteria were isolated of which 52 bacteria were categorized as ESKAPE pathogens. The most common bacteria isolated were 25% (n = 19/76) P. aeruginosa and 17.1% S. aureus. A high level of antibiotic resistance was shown in all ESKAPE and non-ESKAPE pathogens. The Gram-negative bacteria of ESKAPE pathogens were further analyzed comparing 3rd generation cephalosporin and carbapenems resistance patterns. A. baumannii showed the highest resistance towards 3rd generation cephalosporin and carbapenems. In addition, P. aeruginosa showed high resistance to 3rd generation cephalosporins with 89.5% resistance, with E. coli showing high resistance to carbapenems with 50.0% resistance. The burden of ESKAPE pathogens is high in pus swabs obtained from admitted patients at Muhimbili National Hospital. The results showed high antibiotic resistance within ESKAPE and non-ESKAPE pathogens including the “last resort” antibiotics: 3rd generation cephalosporin and carbapenems.


Background
Antimicrobial resistance (AMR) is a global public threat to the treatment and cure of infectious diseases. A problem that arose from over-prescription of drugs by poor administration and inefective therapy, and the abusive use of antimicrobials without the supervision of medical personnel [1]. Te World Health Organization (WHO) categorized AMR as the ffth most urgent threat to global health, ranking above high-threat pathogens including hemorrhagic fevers, highlighting the severity of AMR in the world [2]. With the growing number of multidrug resistant (MDR) bacteria, the Infectious Diseases Society of America (IDSA) [3] termed the most notorious antibiotic-resistant bacteria in an acronym, ESKAPE: E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, and Enterobacter species. Te ESKAPE pathogen cause infections in high-risk groups of people which include immunocompromised, hospitalized, and unwell patients resulting in life-threatening infections, prolonging hospital stays, morbidity, and mortality, and increasing fnancial burden [3][4][5].
Te ESKAPE pathogens have a high potential for developing and acquiring drug resistance mechanisms: drug inactivation, modifcation of drug binding sites, decreased intracellular drug accumulation, and redefning paradigms in pathogenesis, transmission, and treatment [6,7]. Te listed ESKAPE pathogens have an increased occurrence of developing resistance towards 3 rd generation cephalosporins and carbapenems. Te dissemination of resistant bacteria within communities is an emerging global threat that deserves special attention [8][9][10].
Multiple studies report ESKAPE pathogens contributing to a large number of bacteria isolated from various specimens of diferent age-group patients admitted within different departments within the hospital. Benko et al., (2020) described 72.22% (n = 4974/6887) prevalence of ESKAPE pathogens isolated in the emergency department. Klebsiella spp. has low susceptibility to 3 rd generation cephalosporins and beta-lactams except for carbapenems whilst A. baumannii demonstrated high resistance to all antibiotics including carbapenems [11]. Previous surveillance in an intensive care unit (ICU) in Mexico also describes high resistance of ESKAPE pathogens to 3 rd generation cephalosporin and carbapenem, particularly highlighting the alarming rates of resistant A. baumannii. In addition, the study reported E. coli as a frequently isolated bacteria with high associated antibiotic resistance [4,12].
Data on the categorization of ESKAPE pathogens in Tanzania are fnite, and with the burden of emerging antibiotic-resistant organisms isolated in patients causing high morbidity and mortality rates, it is crucial to understand the degree of ESKAPE pathogens' contribution to patient infection [13,14]. Tus, the study aimed to describe the extent and resistance patterns of ESKAPE pathogens isolated in pus swabs from patients admitted to Muhimbili National Hospital.

Study Design and Duration.
A retrospective cross-sectional data analysis was conducted during the 2 nd week of August 2019 at MNH, Dar es Salaam, Tanzania. A total of 75 admitted patients with open wounds and surgical site infections (SSI) were recruited. Patient fles were reviewed to collect microbiological laboratory data and relevant patient data.

Study Population. All patients admitted with an open
wound from diferent wards were subjected to bacteriological processing at the Central Pathology Laboratory (CPL) for pus swab culture. A total of 75 patient pus swabs specimen data were reviewed which included swabs from the skin, traumatic wounds, surgical wounds, burns, nose, eyes, and ear discharge.

Data Collection.
A convenience sampling methodology was implemented, and data were retrieved from in-patient fles. Available patient data: age, sex, and admitted ward, and microbiological data: organism identifcation and antibiotic susceptibility test, were obtained.

Ethical
Clearance. Ethical clearance number DA. 287/ 298/01A/ and permission MNH/TRCU/IRB/permission/ 2019/117 to conduct the study were obtained from the ethical committee of the Muhimbili University of Health and Allied Sciences and Muhimbili National Hospital, accordingly. Patient identifcation data were deidentifed before data analysis to maintain utmost patient confdentiality.

Laboratory
Procedures. All pus swabs were collected aseptically using sterile swabs on clean viable tissue, avoiding pus or necrotic tissue after initial cleaning of the wound with normal saline to remove normal fora. Trained infection prevention and control personnel were responsible to collect the specimen. Specimens collected were transported by Stuart Transport Media within 2 hours of collection. Pus swabs were inoculated on sheep Blood agar (BA) and MacConkey agar (MCA) were incubated at 37°C in ambient air for 24 hours. Colony morphology, gramstain, and conventional biochemical tests were applied to identify isolated bacteria. Biochemical tests for Grampositive bacteria: catalase and coagulase tests; for Gramnegative bacteria: glucose/sucrose fermentation, hydrogen sulfde production, indole production, urease production, citrate utilization, and motility. An API20E (analytical profle index test) was used for Gram-negative bacteria which failed to be identifed using conventional methodology.

Results
Te study population included 75 admitted patients presenting with an open wound and SSI subjected to pus swabs. Te median age of the study was 32 years (IQR; 1 day-73 years). Tere was an equal number of males and females accounting in the study population with 36 males and 36 females. Te wards ranged from surgical ICU, surgical wards, and pediatric ICUs and wards. A total of 76 clinically signifcant bacteria were isolated, with 68.4% (n � 52/76) ESKAPE pathogens as shown in Figure 1.

Resistance Patterns.
Among the antibiotic groups tested against the pathogens isolated A. baumannii displayed the highest resistance, with no isolate sensitive to antibiotics such as cephalosporins, aminoglycoside, and sulfonamides. A. baumannii isolated also displayed the highest resistance to carbapenems among the ESKAPE pathogens.
A.baumannii (6)   K. pneumoniae ranked the second bacteria among the ESKAPE pathogens showing high resistance to antibiotics cephalosporin and sulfamides with 11.1% sensitivity shown in Table 1.
In the Gram-positive ESKAPE pathogens, S. aureus was comparatively sensitive to the antibiotic groups. However, it showed resistance to cephalosporin with 38.5% sensitivity. Te highest sensitivity was shown in carbapenems and fuoroquinolones.
Among the species isolated not part of the ESKAPE pathogens, E. coli displayed high resistance towards all antibiotic groups listed, with the highest resistance to β-lactam combination, sulfonamides, and fuoroquinolones: 12.5% sensitivity. Proteus spp. also showed a high level of resistance to the antibiotic groups with 0.0% sensitivity for antibiotics aminoglycoside; however, sensitivity towards carbapenems and sulfonamides was displayed.
Resistance patterns of the Gram-negative bacteria among the ESKAPE pathogens list were further scrutinized comparing the resistance towards the "last resort" antibiotics used in patient care which include 3 rd generation cephalosporin and carbapenems. E. coli was also included in the analysis due to the high resistance levels shown in Table 1.
Comparing the 3 rd generation cephalosporins and carbapenems resistance patterns; 3 rd generation cephalosporin resistance was relatively high for all Gram-negative ESKAPE pathogens and E. coli showed in Figures 3 and 4

.
A. baumannii showed the highest resistance towards 3 rd generation cephalosporin and carbapenems compared to other pathogens included, with no isolate sensitive to 3 rd generation cephalosporin and 1 isolate sensitive to carbapenems. Tis is followed by E. coli showing high resistance towards 3 rd generation cephalosporin with 25.0% sensitivity and carbapenems with 50.0% sensitivity.
P. aeruginosa was the most frequent bacteria isolated, comparatively showing high resistance towards 3 rd generation cephalosporin but high sensitivity towards carbapenems. In addition, Enterobacter spp. were 100.0% carbapenem sensitive.
Bacteria commonly reported in wound infection and SSI were P. aeruginosa, K. pneumoniae, S. aureus, E. coli, A. baumannii, Proteus spp. [15,17]. Tis study isolated uncommon bacteria such as Aeromonas hydrophilia and Streptococcus pyogenes which infrequently cause human infections. Te isolation of S. pyogenes is infrequently isolated in human infections, mostly isolated in neonates. Surveillance of AMR in hospitals in Tanzania revealed 0.0005% (n � 4/7617) of S. pyogenes were isolated, terming the bacteria uncommon [18].
Surgical wards had the highest number of pathogens isolated with 69.7% (n � 53/76), with the most frequent bacteria isolated P. aeruginosa 24.5% (n � 13/53). It is recorded that the burden of wound infections is high in hospitalized patients in the ICU compared to regular wards [19][20][21]. However, the high number of bacteria isolated from surgical wards can be attributed to overcrowding and understafng in hospitals, resulting in poor infection control practices [22]. Tis suggests the presence of nosocomial infection within the surgical ward.
Te study also reported a high level of resistance among pathogens isolated from pus swabs of patients admitted to MNH. Te highest resistance among all the pathogens isolated in the study was displayed by A. baumannii. Among the remaining ESKAPE pathogens, high resistance was displayed in K. pneumoniae and P. aeruginosa. For the non-ESKAPE pathogens, high resistance was prominent in E. coli isolates. A total of 17.1% (n = 13/76) of S. aureus were isolated in the study, with 7.7% (n = 1/13) categorized as MRSA and the remaining MSSA. Tere is a low reported MRSA prevalence Table 1: Sensitivity patterns of bacteria isolate in percentage. Carbapenem (imipenem/meropenem), β-lactam combination (piperacillintazobactam), cephalosporin (ceftazidime/cefotaxime or ceftriaxone for gram-positive bacteria), aminoglycoside (amikacin/kanamycin), Sulfonamides (cotrimoxazole), fuoroquinolones (ciprofoxacin), and penicillin.  in Tanzania which corresponds with the results of this study. In 2012, a study aimed to characterized MRSA from wound infections from a total of 26.7% (n = 160/600) S. aureus, of which 15% were MRSA [23]. In 2018, a study aimed to determine the nasal carriage of MRSA among healthcare workers reporting the overall prevalence of MRSA carriers was 15.6% (n = 59/379) [24]. In contrast to other East African countries, Tanzania has low levels of reported MRSA carriers isolation from clinical specimen [17,22,25,26].
Resistance patterns of Gram-negative bacteria of ESKAPE pathogens towards 3 rd generation cephalosporins and carbapenems were further analyzed. A. baumannii showed the highest resistance to both 3 rd generation cephalosporins (0.0% sensitivity) and carbapenems (16.7% sensitivity) among all the bacteria compared. E. coli also showed high resistance to the antibiotic groups: 3 rd generation cephalosporin (25.0% sensitivity) and carbapenems (50.0% sensitivity). Te fndings of this study accord with  other studies identifying A. baumannii and E. coli as being notorious for building carbapenem resistance [15,17]. According to other studies, a signifcant proportion of A. baumannii infections are reported as nosocomial infections with the ability to develop antibiotic resistance [4,20,21]. Enterobacter spp. and P. aeruginosa were comparatively sensitive to carbapenems; however, showed resistance towards the 3 rd generation of cephalosporins. P. aeruginosa resistance to 3 rd generation cephalosporin is attributed to the intrinsic resistance present in the bacteria causing high production of extended-spectrum β-lactamases (ESBLs) [8,27,28]. Te low carbapenem resistance in P. aeruginosa also concurs with other studies isolated from SSI [13,15].

Study Limitation. Determination of ESKAPE pathogens into ESBLs-producing Enterobacteriaceae (ESBL-PE) and
Carbapenemase producing Enterobacteriaceae (CPE) would have provided further information aiding in categorizing appropriate antibiograms for pathogens isolated from the pus swabs. In addition, the small sample size limits the study for generalization and recommendation. Te MDR bacterial infection caused by ESKAPE pathogens can vary depending on the duration of hospital stays, clinical indication, previous antibiotic use, previous MDR bacterial infection, and immunocompromised status of an individual which was not collected.

Conclusion
Te burden of ESKAPE pathogens is very high in pus swabs obtained from admitted patients at MNH with the most frequent bacteria isolated being P. aeruginosa at 25.0% (n � 19/76) followed by S. aureus at 17.1% (n � 13/76). Among the non-ESKAPE pathogens, 15.7% (n � 12/76) Proteus spp. and 10.5% (n � 8/76) E. coli were the most common pathogens isolated. Te fndings of high resistance towards the "last resort" antibiotic groups: 3 rd generation and carbapenems; urge the surveillance of ESKAPE pathogens, including E. coli and stress the importance of laboratory tests to further categorize the bacteria into ESBL-PE and CPE, providing grounds for antibiotic stewardship to guide clinicians for appropriate prescription. Tis would limit the acceleration of MDR bacteria among admitted patients, reducing hospital stays and encouraging fast recovery. Te study fndings also recommend further research into diferent innovative and inventive techniques in combating the crisis of AMR, which are efective and efcient [29][30][31].

Data Availability
All data analyzed during the study are included in this published article.

Conflicts of Interest
Te authors declare that there are no conficts of interest.