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. 2022 May 16;2(5):e0000417. doi: 10.1371/journal.pgph.0000417

Antibiotic-resistant pathogenic bacterial isolates from patients attending the outpatient department of university of Cape Coast hospital, Ghana: A retrospective study between 2013–2015

Kwame Kumi Asare 1,*, Samuel Amoah 2, Cornelius Agyeman Coomson Jr 1, Cecil Banson 2, Derrick Yaro 1, Jennifer Mbata 3, Rudolf Aaron Arthur 2, Peter Bilatam Mayeem 4, Justice Afrifa 5, Felicity Bentsi-Enchill 3, Yeboah Kwaku Opoku 3
Editor: Kwabena Obeng Duedu6
PMCID: PMC10021532  PMID: 36962199

Abstract

Uropathogenic Escherichia coli (E. coli) is an important urinary tract infection (UTI) that has been associated with both complicated and uncomplicated disease conditions. The global emergence of multiple drug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) is of public health concern as the resistance limits the current treatment options. The objective of this study was to analyze the antibiotic-resistant patterns among the uropathogenic E. coli isolates at the University of Cape Coast (UCC) hospital between 2013 and 2015 as baseline data to understand the current antibiotic resistance situation within UCC and its environs. A retrospective cross-sectional study of bacteria isolates at UCC hospital from January 2013 to December 2015 were analyzed. A standard biochemical and antibiotic susceptibility tests were performed using Kirby-Bauer NCCLs modified disc diffusion technique. The network of interaction between pathogenic isolates and antibiotic resistance was performed using Cytoscape software. Statistical significance was tested using ANOVA and one-sample Wilcoxon test. The overall E. coli prevalence was 15.76% (32/203); females had the highest infection of 17.33% (26/150) compared to male subjects who had 11.32% (6/53) out of all the pathogenic infections. The E. coli prevalence among the age categories were 2/21 (9.52%), 27/154 (17.53%) and 4/21 (19.05%) among ≤20 years, 21–40 years and 41–60 years respectively. The isolated resistant pathogens exhibited different antibiotic resistance patterns. An interaction network of nodes connecting to other nodes indicating positive correlations between the pathogens and antibiotic resistance was established. Escherichia coli, Citrobacter spp, Klebsiella spp among other isolated pathogens formed higher centrality in the network of interaction with antibiotic resistance. The individual E. coli isolates showed a significant difference in the mean ± SD (95% CI) pattern of antibiotic resistance, 2.409±1.205 (1.828–2.990), χ2 = 36.68, p<0.0001. In conclusion, the study reports the interaction of E. coli isolates at UCC hospital and its antibiotic-resistant status between 2013 and 2015. This data forms the baseline information for assessing the current antibiotic status in UCC and its environs.

Introduction

Antimicrobial resistance is a threat to health. The increasing reports of bacteria resistance to antibiotics limit the treatment options and render commonly encountered bacterial infections such as urinary tract infections (UTIs) difficult to treat. UTIs are invasive or progressive bacterial infections that have exceeded their pathogenic threshold of 105 colony-forming unit (CFU)/mL [1, 2]. UTIs are the third most common bacterial disease associated with the frequent cause of morbidity in both outpatients and hospitalization besides respiratory and gastrointestinal infection [3, 4]. Escherichia coli, Citrobacter species, Staphylococcus aureus, Staphylococcus faecalis, Proteus mirabilis, Klebsiella species, Streptococcus species and others are the most frequent cause of UTIs in both developing and developed countries [5, 6]. The adherence of uropathogens to the epithelial is followed by the production of bacteria toxins, which further stimulates an inflammatory response resulting in cell destruction [7, 8]. The cardinal symptoms of bacteria colonization of the urinary tract infections range from cystitis, urethritis, pyelonephritis, hematuria, dysuria, cloudy urine and nocturnal enuresis [9, 10]. Thus, compromising the host immune defence mechanisms and inducing pathogenicity and virulence by pathogenic bacteria [11].

The uropathogenic E. coli (UPEC) is the most frequently isolated pathogen involved in 25–50% of complicated and more than 70% of uncomplicated UTI cases [12, 13]. The UPEC consist of only a selected number of E. coli strains that successfully survive, colonize and cause infection in the urinary tract [14]. The UPEC produces virulence factors (VFs) and acquires antibiotic resistance genes required for biofilm formation and nutrient or iron acquisition, invasion of host-cell cytotoxicity or host immune defence and resistance to antibiotic treatment [15–17]. These E. coli virulence factors are essential for the bacteria to survive in varying environmental conditions, metabolize and use several carbon sources for facultatively anaerobic metabolism [18]. About 20% of female vaginal microbiota constitute UPEC and it is associated with aerobic vaginitis [19, 20].

Improper stewardship, the increased use of unprescribed antibiotics, improper prescription of broad-spectrum antibiotics and their overuse are among the contributing factors for the rapid emergence of antibiotic resistance. The prevalences of E. coli resistance in high-income countries are Trimethoprim (53.4%), co-amoxiclav (23.6%), ampicillin (8.2%), ciprofloxacin (2.1%) while that of low and middle-income countries are ampicillin (79.8%), co-amoxiclav (60.3%), and ciprofloxacin (26.8%). Antibiotic resistance surveillance in low and middle-income countries lacks. E. coli resistance to co-trimoxazole (77%), ampicillin (93%) and gentamicin (29%) in several reported cases. In Ghana, E. coli antibiotic resistance to Amoxicillin, Ampicillin, Cotrimoxazole, Tetracycline, Chloramphenicol, Cefuroxime, Gentamicin, Amikacin, Cefotaxime, and Ciprofloxacin are 100%, 86.8%, 81.6%, 73.6%, 34.42%, 22.6%, 12.74%, 6.97% and 5.7% respectively.

The emergence of antibiotic resistance among the UPEC isolates is a challenge as it substantially influences treatment options [21, 22]. The global emergence and increase of multidrug-resistant (MDR) E. coli are of prime importance for the management of UPEC [23]. The ESBLs produced by UPEC is able to hydrolyze broad-spectrum cephalosporins and other monobactams [24, 25]. The UPEC antibiotic resistance to cotrimoxazole, ampicillin, nitrofurantoin and fluoroquinolones such as ciprofloxacin and levofloxacin is of major concern [26, 27]. The wide range of UPEC resistance to all classes of antibiotics limits the alternative treatment options [28].

The UPEC antibiotic resistance is not only a threat to nosocomial and healthcare-associated infections but also community infections [29, 30]. The trend and risk factors associated with antibiotic-resistant pathogens associated with UTIs require constant review among bacteria isolates [31, 32]. Such knowledge is essential for policies implementation and the proper usage of antibiotic drugs to combat MDR in UTIs. The study analyzed the antibiotic resistance patterns among pathogenic bacterial isolates from the outpatient department (OPD) at the University of Cape Coast (UCC) hospital between 2013 and 2015 to serve as baseline data to understand the current antibiotic resistance situation within UCC and its immediate environs. The isolated pathogens and their antibiotic-resistant status were visualized using the network of interactions to understand the magnitude and the trends of antibiotic resistance among species of pathogenic bacterial isolates.

Materials & methods

Study area

A retrospective cross-sectional study of clinical bacteria culture and isolates carried out using the Microbiology Laboratory register records at the University of Cape Coast hospital from January 2013 to December 2015 were analyzed. The university lies between 50 8’ 10” N, 10 17’ 56” W to NE and 50 5’ 51” N, 10 16’ 43”W to SE. The hospital caters for the health needs of students and staff of the university as well as individuals in the surrounding communities. The hospital is situated at the main entrance to the university campus, which is about 160 meters from the shores of the Gulf of Guinea.

Ethical approval

Ethical approval from the Department of Biomedical Sciences Research Board, University of Cape Coast (DBRB/17/0432) was obtained before the study and permission from the Medical Administrative Committee of the University of Cape Coast Hospital. All data obtained from Laboratory records were anonymous.

Clinical sample collection and identification of pathogens

Convenience sampling was used for the selection of samples. The study samples included high vaginal swabs, blood samples, urethral swabs, urine samples, stool samples, cerebrospinal fluids samples, ear swabs and wound samples collected at the University of Cape Coast hospital. The patients’ demographic characteristics such as age and sex were also recorded from the registrar. Blood agar (Sigma-Aldrich) and MacConkey agar (Sigma-Aldrich) were used for the isolation of the pathogens. The incubation conditions were 37oC aerobically overnight. The isolates were identified based on colony appearance, Gram stain, catalase-positive, and oxidase negative isolates were further examined.

The bacteria isolates analyzed in this study were obtained using the standard traditional biochemical tests and selective media such as Endo agar (Sigma-Aldrich), MacConkey broth, Simmons citrate agar (Sigma-Aldrich), catalase (Sigma-Aldrich), coagulase (Sigma-Aldrich), oxidase (Sigma-Aldrich), sugar fermentation [Triple sugar iron (TSI) agar] (Sigma-Aldrich), indole (Sigma-Aldrich), citrate utilization (Sigma-Aldrich), urease production (Sigma-Aldrich), and motility tests were used [33]. The Neisseria gonorrhoeae confirmatory test was performed using Identicult-Neisseria (Adams Scientific, USA) test [34]. The test depends on beta-galactosidase, gamma-glutamylaminopeptidase and prolyl-hydroxypropyl aminopeptidase and rapid carbohydrate utilization test (RCUT) of clinical isolates of oxidase-positive Gram-negative diplococci. E. coli confirmatory test was performed using Sorbitol-MacConkey (SMAC) agar with E. coli O157 antiserum or latex reagents (O157 antibody-coated latex and control latex) based on the manufacturer-recommended procedures.

Candida species detection

Candida spp analyzed in this study was obtained by cultivating suspected specimens on Sabouraud glucose agar (SGA) and subsequently confirmed morphologically using assimilation/fermentation methods or cultured Corn meal-Tween 80 agar (CTA)

Antibiotic Susceptibility Test (AST)

Antimicrobial susceptibility was done by Kirby–Bauer disc diffusion method on Mueller–Hinton agar. Briefly, the bacteria turbidity was measured by comparing the normal saline emulsified pure colonies and 0.5 McFarland solution. The susceptibility of bacteria to Ampicillin (Amp) (10μg), Gentamicin (GEN) (10μg), Cotrimoxazole (COT) (25μg), Cefuroxime (CRX) (30μg), Erythromycin (ERY) (5μg) and Amikacin (AMK) (30μg), amoxicillin/clavulanic acid (AMC: 30 μg), ceftazidime (CAZ) (30 μg), imipenem (IPM) (10 μg), Cefixime (CXM) (30μg), Cefotaxime (CTX) (30μg), Penicillin (PEN) (10 IU), Cloxacillin (CXC) (5μg), ertapenem (ETP) (10 μg), meropenem (MEM) (10 μg), tetracycline (TET) (30 μg), trimethoprim-sulfamethoxazole (SXT) (30μg), gentamicin (GEN) (10μg), nalidixic acid (NAL) (30 μg), ciprofloxacin (CIP) (5 μg), chloramphenicol (CHL) (30 μg), aztreonam (AZT) (15 μg), piperacillin/tazobactam (TZP) (100/10 μg), fosfomycin (FOF) (200 μg), and colistin (CST) (10 μg) were examined using Mueller-Hinton agar antibiotic diffusion technique (Kirby-Bauer NCCLS modified disc diffusion technique). CTX, AMC and CAZ antibiotic discs for double-disk synergy test of ESBLs were also performed. The isolated bacteria were plated on a dry Mueller Hinton agar plate with appropriate antimicrobial-impregnated disks and cultured overnight at 35°C. The antibiotic inhibition zones were measured from the centre to the distinct edges of antibiotic inhibition zones using a ruler with E. coli ATCC 25922 strain as a control. The AST discs were obtained from Becton Dickinson (BD, Sparks, MD, USA).

Diagrammatic presentation of relationships between pathogenic bacterial isolates and antibiotic resistance

The network tool (Cytoscape) reduces the underlying complexity in pathogens and antibiotic resistance association through the diagrammatic representation for concepts and relationships. The analysis assessed the characteristics of the isolated pathogens and antibiotic resistance patterns through metrics measurements such as degree, clustering coefficient, shortest paths, centrality, density. The network analysis provides visualizing of the direct association between pathogens and their antibiotic resistance status. It also estimates pathogens and their trends of antibiotic resistance.

Statistical analysis

The data was entered, validated and analyzed using Excel 2016 (Microsoft Corporation) and GraphPad Prism 9.0.2 software. The network of the interaction of isolates and antibiotic resistance were performed using Cytoscape version 3.8.2 software. The statistical significance was tested using ANOVA, and a one-sample Wilcoxon test at p = 0.05.

Result

Demographic characteristics of the patients

A total of 1515 samples were cultured between the years 2013–2015 at the UCC hospital Laboratory with a slightly predominant female prevalence of 51.33%, 69.20% and 63.36% for 2013, 2014 and 2015 respectively. The median (95% CI) for the age 21–40 years for 2013 was 26 (25–28), 2014 was 26 (25–27) and 2015 was 25 (25–26). High vaginal swab (40.64%, 39.45% & 36.64%) and urine (37.97%, 58.85% & 55.22%) were the most frequent sample collected from 2013–2015 (Table 1). The trends of pathogens isolation were high among females compared to males and the median age between subjects ranged between 20 and 35 years (S1 Table).

Table 1. Demographic characteristics of the patients.

2013 2014 2015
Sex n/N (%)
Male 91/187 (48.66) 235/763 (30.80) 207/565 (36.64)
Female 96/187 (51.33) 528/763 (69.20) 358/565 (63.36)
Age n/median (95% CI)
≤20 years 24/18 (15–20) 131/18 (16–19) 113/17 (12–19)
21–40 years 113/26 (25–28) 513/26 (25–27) 344/25 (25–26)
41–60 years 40/49 (45–50) 88/50 (48–51) 68/49 (48–51)
61–80 years 10/71 (61–75) 31/70 (66–73) 40/69 (67–73)
Isolates n (%)
Pathogenic bacteria 93 (49.73) 223 (29.23) 147 (26.02)
Commensal bacteria 30 (16.04) 113 (14.81) 50 (8.85)
Fungi infection 19 (10.16) 97 (12.71) 72 (12.74)
Specimen n (%)
HVS 76 (40.64) 301 (39.45) 207 (36.64)
Urethral 40 (21.39) 13 (1.70) 46 (8.14)
Urine 71 (37.97) 449 (58.85) 312 (55.22)
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An increase in multiple antibiotic-resistant Escherichia coli infections among female patients between the ages 21–40 years

Among the pathogenic isolates, the overall prevalence of E. coli was 17.33% (26/150) and 11.32% (6/53) for females and males respectively. Among the female patients, the age group 21–40 years recorded the highest burden of the number of isolated pathogens compared to ≤20 years and 41–60 years age groups. The age group 21–40 years had 12 (44.44%) different pathogenic isolates compared to 7 (25.93%) and 8 (29.63%) isolates for the age groups ≤20 and 41–60 years. The E. coli isolates were 2/21 (9.52%), 22/114 (19.30%) and 2/15 (13.33%) among ≤20 years, 21–40 years and 41–60 years respectively. The one-sample Wilcoxon test showed significant difference among the E. coli isolates compared to other pathogenic bacteria W = 28.0, 2.0 (1.0–7.0), p = 0.0156; W = 78.0, median (95% CI), 5.50 (3.0–15.0), p = 0.0005 and W = 36.0, 2.0 (2.0–3.0), p = 0.0078 among ≤20 years, 21–40 years and 41–60 years respectively. The residual plot showed a dynamic variation pattern of bacterial infections across the age of 21–40 years (Fig 1A). The prevalence of E. coli infection isolates from HVS among the age categories ≤20 years, 21–40 years and 41–60 years were 8.33%, 7.55% and 16.67% respectively. Again, a coinfection of 5.66% was recorded for E. coli & Candida spp for the age group 21–40 years (Fig 1B). The pathogenic infections observed among the urine samples collected from female patients across the age categories ≤20 years, 21–40 years and 41–60 years showed a similar pattern to that of HVS infections. The number of pathogenic isolates was very low among the age categories ≤20 years with E. coli infection representing 33.33% of the isolates. The prevalence of E. coli from the urine specimens among the age groups 21–40 years and 41–60 years were 17.39% and 25.0% respectively (Fig 1C). Notably, more pathogenic bacteria isolates were recorded from the HVS (57.25%) and urine (19.85%) specimens compared to the other forms of specimens collected. The one-sample Wilcoxon statistic showed a significant difference in the median (95% CI), 8.0 (7–75.0), W = 21.0, p = 0.0313 among the specimens analysed (Table 1).

Fig 1. An increased urogenital antibiotic-resistant Escherichia coli infection among females between the ages 21–40 years old.

Fig 1

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A. The trends in the bacteria isolate among female patients with age categories ≤ 20 years, 21–40 years and 41–60 years. The residual plot analysis showed changes in the trends of bacteria isolates among the age 21–40 years. B. The frequency of bacteria isolates from different specimen types across the age categories ≤ 20 years, 21–40 years and 41–60 years. C. The interleaved bars showing frequencies of bacteria isolates from high vagina swabs (HVS) samples across the age ≤ 20 years, 21–40 years and 41–60 years.

An increase in multiple antibiotic-resistant Escherichia coli infections among male patients between the ages 21–40 years

Even though the majority of the male samples analysed were from urine (38.05%) followed by urethral swabs (33.63%), the urethral swabs had the largest number of different pathogenic bacteria isolates. S. epidermidis (50.94%), N. gonorrhoeae (15.09%), E. coli (9.43%) and S. saprophyticus (7.55%) were the most dominant pathogenic bacteria isolated from the male urethral. The one-sample Wilcoxon test showed a significant difference among the isolates, median (2.50), W = 55.0, p = 0.002 (Fig 2A). The different specimens collected from the male patients had different pathogenic bacteria infections with variations in their prevalence. A prevalence of 9.43% and 20.0% E. coli isolates were recorded from urethral and urine samples respectively (Fig 2B). The prevalence of E. coli from the urethra among the ages between 21–40 years was 12.5% (5/40) (Fig 2C). E. coli 33.3% (2/6) was only isolated from the urine samples among the ages 41–60 years in the male subjects (Fig 2D).

Fig 2. High isolation of multiple antibiotic-resistant E. coli from urethral swab among male patients between 21–40 years of age.

Fig 2

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A. The frequency of different bacteria isolates from urethral swab samples. 50.94% of the isolates were Staphylococcus epidermidis, 15.09% were Neisseria gonorrhoeae and 9.43% Escherichia coli being the third-highest bacteria isolates from the male urethra. B. Pie charts showing the various % frequencies of bacteria isolated from the different specimens collected from male patients attending UCC hospital. Escherichia coli were only isolated from urethral swabs (9.43%) and urine (20%) among the respective bacteria isolates from each specimen. C. The interleaved bars showing frequencies of bacteria isolates from Urethral swabs samples from male patients across the age categories ≤ 20 years, 21–40 years and 41–60 years. D. The interleaved bars show frequencies of bacteria isolates from urine samples across the age categories 21–40 years and 41–60 years. No bacteria isolates were obtained among the age ≤ 20 years.

The network of interactions of pathogenic bacteria isolates and antibiotic resistance

The isolated resistant pathogens exhibited different antibiotic resistance patterns (S2 Table). To assess the interaction between the pathogenic isolates and antibiotic resistance, an interaction network analysis was performed. The result showed a network of nodes connecting to other nodes indicating positive correlations between these pathogens and their sensitivity to various antibiotics (Fig 3A). The individual pathogenic isolates formed a separate cluster of interactions (edges) with the various antibiotics and there was no direct interaction between the individual isolated pathogens. E. coli, Citrobacter spp, Klebsiella spp and the other isolated pathogens formed higher centrality in the network of interaction with antibiotic resistance (Fig 3B). This indicates the difference in the levels of antibiotic resistance among the individual pathogens. To understand the interactions, the individual E. coli isolates from stool, HVS and urine were further analyzed for their antibiotic resistance patterns. The individual E. coli isolates showed a significant difference in the mean ± SD (95% CI) pattern of antibiotic resistance, 2.409±1.205 (1.828–2.990), χ2 = 36.68, p<0.0001 (Fig 3C). The prevalence of Bactrim (BA), tetracycline (TE), ampicillin-sulbactam (AS), & chloramphenicol (CH) antibiotic resistance were 34.55%, 25.45%, 18.18% & 9.09% among the isolated E. coli. Furthermore, 42.11% vs 42.11%, 35.71 vs 57.14%, 60% vs 0% and 40% vs 50% were recorded as the antibiotic resistance pattern for BA, TE, CH and AS among the E. coli isolated from HVS vs urine specimen (Fig 3D). Type III two-way ANOVA analysis showed that E. coli isolates from stool, HVS and urine exhibit a significant difference in their antibiotic resistance patterns F = 5.399, p = 0.0408.

Fig 3. A unique and separate interactions of bacteria isolates and antibiotic resistance pattern.

Fig 3

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A. Bacteria isolates interaction network, in which nodes represent different associations with antibiotic resistance and bacteria isolates. B. Zoom-in of the interaction networks showing individual bacteria isolates forms a separate and unique interaction. The Escherichia coli and Citrobacter species showed interesting interaction networks. C. The different antibiotic resistance patterns of Escherichia coli isolates from the stool, High vaginal swabs (HVS) and urine specimens. The bar graph is the mean E. coli isolates with similar antibiotic resistance pattern and each dot represents the mean of the individual antibiotics. D. The stacked bars show the various antibiotics and the prevalence of resistant Escherichia coli isolates from stool, HVS and urine. Bactrim (BA), tetracycline (TE), ampicillin-sulbactam (AS), clindamycin (CL), ofloxacin (OF), lincomycin (LZ) and chloramphenicol (CH).

Discussion

Multiple drug resistance (MDR) pathogenic bacteria associated with severe morbidity in UTIs is a major global health concern [35, 36]. Antibiotic resistance and rapid spread of aminoglycosides and the lactams such as cephalosporins and fluoroquinolone among uropathogenic bacteria undermines the clinical management of the infection and result in a poor prognosis [37, 38]. The development of resistance to the third-generation cephalosporins and nitrofurantoin are a major cause of prolonging hospitalization among infected patients and limiting bacteria treatment options [39, 40]. The antibiotic-resistant uropathogens employ several resistance mechanisms including the expressions of β-lactamases, TEM-, SHV-, CTX-M-, and OXA-type resistance genes responsible for ESBLs [41, 42]. ESBLs producing bacteria differs from patients, clinical and geographical settings even though the main source of ESBL producing bacteria are nosocomial [43]. This study reports on uropathogenic E. coli infections among patients visiting the University of Cape Coast hospital from January 2013 to December 2015 and their antibiotic-resistant patterns.

The study showed that E. coli significantly colonized the female vaginal and male urethral compared to other pathogens and the majority of UPEC colonization were observed among the 21–40 years category. Studies have documented the association of E. coli colonization of the vagina with very low birth weight and preterm delivery [44, 45]. Also, the vagina and male urethra colonization of the sperm agglutinating strain of E. coli induces infertility and treatment with antibiotics improves the quality of semen and increases fertility among females [46, 47]. Coincidentally, 21–40 years, which happens to be the fertile stage happens to be the most frequently affected by E. coli infection.

E. coli adhere and colonize the uroepithelial cells, resist urinary stream elimination, and avoid specific immune reactions [48, 49]. E. coli infections cause disease by stimulating cytotoxic activities and local and systemic inflammatory responses [50, 51]. The stimulated local inflammation causes the inflex of granulocytes, macrophages, and monocytes into the tissue [52]. This causes the secretion of proinflammatory cytokines such as IL-1, IL-6, IFN-γ, TNF-ɑ, and infiltration of polymorphonuclear cells to activate an acute-phase response [53].

Uropathogenic E. coli is reported to cause most of the clinically important UTIs globally with a considerably high resistance rate to amoxicillin, tetracycline, and trimethoprim/sulfamethoxazole [54]. The simultaneous resistance of E. coli to several antibiotics has become a worldwide problem. The network analysis showed unique interactions between individual pathogenic isolates and their resistance to the various antibiotics. Ciprofloxacin (Cipro), ofloxacin (Floxin), trimethoprim-sulfamethoxazole (TMP-SMX; Bactrim, Septra), and nitrofurantoin (Furadantin) antibiotics have been used for the treatment and eradication of UTIs [55]. However, treatment failures of E. coli to TMP-SMX had been associated with resistance to these antibiotics [56, 57].

The Surveillance Network (TSN) database has previously reported 38%, 17%, 0.8% and 1.9–2.5% E. coli resistance respectively to ampicillin, TMP-SMX, nitrofurantoin, and fluoroquinolones [58, 59]. In this study, E. coli resistance to Bactrim (BA) was the highest followed by tetracycline and ampicillin-sulbactam (AS) or clindamycin (CL). Again, these UTI isolates exhibited multidrug resistance. The fluoroquinolone antibiotics have been used for the treatment of resistant E. coli in uncomplicated UTIs as a first-line drug in communities that have reported 10–20% resistance to TMP-SMX [60]. However, several studies have reported resistance to ciprofloxacin and ofloxacin [61]. In this study, we also report an observed E. coli resistance to ciprofloxacin and ofloxacin.

Antibiotic-resistant E. coli isolated from the urine or the urinary tract is of clinical significance due to a high concentration of antibiotics found in the urine samples [56, 62]. The resistance of TMP-SMX has been steadily increasing in developing countries due to the easy transfer of high-level plasmid encoding the resistance genes for both trimethoprim and sulfamethoxazole among gram-negative urinary tract infections [16, 63]. The TMP-SMX resistance had been predicted to emerge rapidly using mathematical modelling even with very small quantities of resistance genes and plasmids [64]. Similarly, the continuous use of these antibiotics in a population is directly related to the persistence of resistance in an environment [65].

The decrease in the first-line antibiotics efficacy, increased hospitalization, morbidity, and mortality is a global public health concern [66]. There is a limitation in quantifying the extent of antibiotic resistance as there is not enough information on the efficacy of antibiotic drugs for the treatment of E. coli infections [67]. Currently, the status and the severity of UTIs in the Central region of Ghana remains unknown. Thus, it is important to understand the severity of the UTIs, virulence factors (VFs) in UPEC isolates, and the risk factors that influence antibiotic resistance in the central region of Ghana. This retrospective study serves as the baseline information to assess current antibiotic resistance status in the University of Cape Coast and its environs in the Central region of Ghana.

The limitation of this study is that the results reported in this manuscript do not reflect the current UTIs and antibiotic resistance status as the pathogenic infections pattern, diversity and treatment regimens may have changed with time. However, this information is important to understand the basis for the evolution of ESBL and MDR antibiotic resistance among the UPEC.

In inclusion, this retrospective study reports the antibiotic resistance status of E. coli isolates from the urinary tract infection. We also report on the pattern of pathogens associated with UTIs, the interaction of the isolates and the various antibiotic resistances among patients visiting the UCC hospital.

Supporting information

S1 Table. The patterns of isolation of resistant pathogens among males and females at the UCC hospital department.

(XLS)

Click here for additional data file. (33KB, xls)
S2 Table. The antibiotics zone of inhibitions among the resistant pathogens isolated from 2013 to 2015.

(XLS)

Click here for additional data file. (37.5KB, xls)

Acknowledgments

We thank the Hospital administration, staff and laboratory technologists of the University of Cape Coast hospital, Ghana for their assistance during the data collection.

Data Availability

The authors confirm that the data supporting the findings of this research are available within the article.

Funding Statement

The author(s) received no specific funding for this work.

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PLOS Glob Public Health. doi: 10.1371/journal.pgph.0000417.r001

Decision Letter 0

Kwabena Obeng Duedu, Julia Robinson

10 Nov 2021

PGPH-D-21-00765

Antibiotic Resistance among Uropathogenic Escherichia coli (UPEC) isolated from patients attending the University of Cape Coast hospital: Retrospective study between 2013-2015

PLOS Global Public Health

Dear Dr. Asare,

Thank you for submitting your manuscript to PLOS Global Public Health. After careful consideration, we feel that it has merit but does not fully meet PLOS Global Public Health’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Dec 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at globalpubhealth@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pgph/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

  • A rebuttal letter that responds to each point raised by the editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

  • A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

  • An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

We look forward to receiving your revised manuscript.

Kind regards,

Kwabena Obeng Duedu, PhD

Academic Editor

PLOS Global Public Health

Journal Requirements:

1. In your ethics statement in the Methods section and in the online submission form, please provide additional information about the data used in your retrospective study. Specifically, please ensure that you have discussed whether all data from the Microbiology Laboratory register records were fully anonymized before you accessed them.

Additional Editor Comments (if provided):

Revise the discussion and ensure results are adequately discussed.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This study is very essential as it reports the frequencies and prevalence of bacterial DR isolates in urogenital specimens in the Central region of Ghana, particularly, in hospital attendees in the UCC Hospital. The use of cytoscape in presentations of data was interesting. However, the figures were not clear (especially Fig 2B, 3A and B) and it will be better if clear single figures are presented for all the images.

Abstract

1. Introduction: separate spectrum from β-lactamase

2. Results: The E. coli prevalence among the age categories were 2/21 (9.52%), 22/114 (19.30%) and 2/15 (13.33%) among ≤20 years, 21-40 years and 41-60 years respectively, the numerators don’t add up to the 32 infections. It looks as if you presented for only the females as the numerator add up to 26 and the denominator adds up to 150. Please recheck and revise

Introduction

1. … their pathogenic threshold of 105 CFU/mL. Indicate the full form of abbreviation on initial use

2. UTIs are the third most common bacterial disease, indicate the first two most common bacterial diseases. This will enrich the background of the study

3. The mechanism of development of bacterial drug resistance was poor explained. Please explain into details mechanisms by which bacteria may become insensitive to antibiotics. The molecular basis was provided but there are human behaviors that encourage bacterial drug resistance.

4. Additionally, review global, continental and local trend of DR and some economic importance of DR regarding bacterial infections

5. The trend and risk factors associated with antibiotic resistance pathogens associated with UTIs require constant, change resistance to resistant

Methods

1. Study clinical sample collection …, authors should indicate the sampling strategy employed in this study whether random or convenient

2. Under the objective of your study was to look at E. coli infections in urosamples. It is not clear why under Clinical sample collection and identification of pathogens, you indicated that pathogens isolated in blood samples, stool samples, cerebrospinal fluids samples, ear swabs and wound samples were also analyzed. Under the same subheading, you indicated that ‘All the samples were spread onto Blood agar (Sigma-Aldrich) and MacConkey agar (Sigma-Aldrich) and incubated at 37°C overnight’. My understanding was that you reviewed laboratory records so its not proper to indicated how the C/S was done in this way. you will have to review that statement to reflect that blood agar (Sigma-Aldrich) and MacConkey agar (Sigma-Aldrich) was used to isolate the pathogen analyzed in this study. They incubation conditions were 37°C aerobically overnight.

3. Under Biochemical Identification, avoid repeating the source of the reagents (Sigma-Aldrich). Revision is required in that regard

4. Change (Triple sugar iron (TSI) agar) to [Triple sugar iron (TSI) agar]

5. MacConkey broth (Sigma-Aldrich), and Simmons citrate agar (Sigma-Aldrich) … no need to insert and between these 2 media while others were to follow

6. As was dine for the other, indicate the source of these reagents; catalase, oxidase, sugar fermentation (Triple sugar iron (TSI) agar), indole, citrate utilization, urease

7. Under Biochemical Identification, revise the section to reflect that, that was the technique used to obtain the isolates used in this study. You may be working in the hospital lab and may be familiar with the protocols for bacteria isolation and identification, you don’t have to indicate as if you purposively isolated these pathogens, remember, this is a retrospective study (analysis of already isolated and identified pathogens)

8. Candida species detection: you can revise this subsection as … Candida spp analyzed in this study was obtained by cultivating suspected specimens on Sabouraud glucose agar (SGA) and subsequently, the isolates were confirmed morphologically using assimilation/fermentation methods or cultured Corn meal-Tween 80 agar (CTA)

9. Under Antibiotic Susceptibility Test (AST), previous comments apply to this section as well

10. Under same subsection, authors have to indicate the source of AST discs used to obtain the susceptibility pattern described in tis study.

11. Under Antibiotic Susceptibility Test (AST), reference 33 is misplaced. Place it after … (Kirby-Bauer NCCLS modified disc diffusion technique). I don’t even understand what that ref means, the list of antibiotics indicated were what you tested your isolates against in your lab so the reference? Reading Anning et al, Antibiotic susceptibility pattern of Enterobacteriaceae isolated from raw meat and Ghanaian coin currencies at Cape Coast metropolis, Ghana: the public health implication only indicated the AST profile for some organisms. Justify the reference or delete it from the ms.

12. Sources of reagents reported indicated in this study. it was interesting to note that all the reagents used to obtain the isolates analyzed in this study was obtained from the same source, was it designed or coincidental?

13. Please provide ethical approval body and reference number for the study

Results

1. Demographics: Authors should include clinical sources of the patients whose samples were analyzed, namely, OPD, in-patient, ANC etc and discussion the implications of this in relation to public health

2. Table 1: I do not get it why you analyzed bacterial pathogens from other sample sources aside urine, HVS and urethral. Based on the focus of your data, there is no need to include pathogens from other samples. Please revise the manuscript

3. The figures used were good but not very clear. Kindly present clear images in your revised manuscript

4. Figure 3D, please define the abbreviations

5. It will be of interest to include the year-by-year gender and age incidence trends of the isolates and drug resistant isolates

6. Authors should provide the ZoIs of the various pathogens regarding each antibiotic as a supplementary material

Discussion

1. TEM-, SHV-, CTX-M-, and OXA-type are resistance genes, please revise to capture that

2. The results section as well as the discussion section was silent on the gender trend of the DR isolates. Please include them appropriately

3. Authors have to understand that various AST disks may give different ZoIs to the same pathogen, hence phenotypic assessment of DR is not conclusive, therefore inclusion of suggestion of molecular surveillance for DR gene markers in pathogens with high ZoIs is essential. Authors can also review some of the genetic markers associated with bacterial DR. Additionally, authors have to discuss some of the reported prevalence of bacterial DR globally, continentally and in some other parts of Ghana.

Reviewer #2: The manuscript entitled “Antibiotic Resistance among Uropathogenic Escherichia coli (UPEC) isolated from patients attending the University of Cape Coast hospital: Retrospective study between 2013-2015” submitted by Asare KK and co-authors describes a retrospective study on microbial pathogens and antibiotic resistance of bacteria isolated from patients attending a university hospital in Ghana during the years 2013-2015. The authors analyzed the data available in the hospital’s registry including demographic (age, gender), biological (biological samples) and microbiological data (pathogen isolated and antibiotic resistance). Overall, the manuscript suffers from several drawbacks that make it unsuitable for publication as it is. Below are specific comments on the content and the quality of the manuscript.

1. The title of the manuscript does not reflect its content. The title mentions a focus on UPEC, while in the results section the authors present an analysis of all the bacterial and Candida infections found in the different biological samples during the study period. I suggest that the authors (i) keep the title and conduct an analysis clearly focused on UPEC isolates found during the study period, or (ii) change the title to more broadly include all the bacterial pathogens isolated.

2. The English level of the manuscript is poor with too many grammar and syntax errors. The manuscript should be combed through by a writer fluent in English to bring it to a standard suitable for a scientific journal.

3. The analysis of bacterial pathogens isolated during the study period is poorly structured with all the data lumped together and very difficult to follow. Analyzing the data in males vs in females, then by age groups is a sound approach. However, these different analyses should be clearly separated in order to show the discoveries. I suggest the authors to divide this analysis to different subsections, each dealing which an aspect, presenting the particular findings and ending with a conclusion.

4. The authors analyzed antimicrobial resistance (AMR) by conducting a network of interactions between pathogenic bacteria and antibiotic resistance. There are several concerns regarding this analysis. Firstly, the methodology of this analysis is not detailed at all in the material and methods section. Secondly, the goals of this analysis is not clearly mentioned by the authors anywhere in the manuscript. Thirdly, the authors stated findings without telling neither their meaning nor their significance. For example, in the subsection of the results section entitled “The network of interactions of pathogenic bacteria isolates and antibiotic resistance” the authors states the following: “The result showed a network of nodes connecting to other nodes indicating positive correlations between these pathogens and their sensitivity to various antibiotics (Figure 3 A)” (lines 2-4 of the above-mentioned subsection). What does this means in terms of antibiotic susceptibility of the corresponding bacteria to the antibiotic tested? The same question pertains to the following sentence where the autors wrote: “The individual pathogenic isolates formed a separate cluster of interactions (edges) with the various antibiotics and there was no direct interaction between the individual isolated pathogens. E. coli, Citrobacter spp, Klebsiella spp and the other isolated pathogens formed higher centrality in the network of interaction with antibiotic resistance (Figure 3 B).” Given the importance of AMR in public health, I suggest that the authors present a detailed analysis of susceptibility of bacteria (either UPEC or all pathogens) to relevant antibiotics before describing network of interactions while giving sound conclusions.

5. The manuscript would much easier to review if it included line numbers.

Below are additional issue that the authors need to address.

Materials and methods

In the subsection entitled “Clinical sample collection and identification of pathogens”, the authors wrote the following

• “All the samples were spread onto Blood agar (Sigma-Aldrich) and MacConkey agar (Sigma-Aldrich) and incubated at 37°C overnight.” Were the same media used whatever the biological sample processed? Since there were various biological fluids analyzed, please mention the different procedures applied if any.

• “The isolates were identified based on colony appearance, Gram stain, catalase-positive, and oxidase negative isolates were further examined [33].” Details on bacterial identification were not provided. In the subsection that followed, the authors mentioned biochemical methods. Please consistently summarize the methods used to identify bacterial species.

There are several inconsistencies in the subsection entitled: “Antibiotic Susceptibility Test (AST)”

• The first sentence is not clear. Start by mentioning the use of disc diffusion, then that 0.5 McFarland suspension are used, etc.

• The authors did not mention the standard they used for AST: EUCAST? CLSI? Other?

• The authors mentioned Cefuroxime twice, one abbreviated as CRX (third line) and the other as CXM (fourth line).

• The authors mentioned Cefotaxime twice (fifth and sixth lines)

• The authors sometimes used a capital letter to begin the names of the drugs and sometimes a small cap letter; additionally, they wrote the abbreviations and the concentrations of the drugs sometimes separately [e.g. (CTX) (30�g)], sometimes together [e.g. (MEM: 10�g)]. Please use a consistent writing method for these molecules.

• At the end of this subsection, the authors mentioned the use of E. coli ATCC 259122 as a QC strain for AST. Since several antibiotics were tested, what are the other QC strains used for drugs not relevant to E. coli ATCC 25922?

Reviewer #3: Dear Authors, please make sure that you have got line numbers in your manuscript next time, therefore reviewers can point the changes directly by saying the line number.

Abstract.

Please seperate "spectrum and beta-lactamase"

What kind of standard biochemical test are done and why?

please mention "patient"

please write full name of E. coli first time and then shorthen it.

spp is not italic.

results section has to be written again, because authors mentioned the antimicrobial resistance pattern testing, but there is no results saying about it.

Introduction:

"species" not italic.

Please seperate a new paragraph from the sentence "About 20%...."

Please check all "comma" necessary places in the mansucript for example before "but" "which" etc.

Please re-write the last sentence of the "Clinical sample collection and identification of pathogens"

"Staphylococcus epidermidis (50.94%), Neisseria gonorrhoeae (15.09%), Escherichia

coli (9.43%) and Staphylococcus saprophyticus" please shorthened the name of these isolates.

"60.0% vs 0.0% and 40.0%" please delete the comma and 0 if the number is integer.

In results section, there is no data regarding the antimicrobial susseptibility test, please include the results.

Discussion:

ESBL is already abreviated.

Most of the discussion section seems like the review paper, rather than discussing the results of this study.

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Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr Enoch Aninagyei

Reviewer #2: Yes: Yakhya Dieye

Reviewer #3: No

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PLOS Glob Public Health. doi: 10.1371/journal.pgph.0000417.r003

Decision Letter 1

Kwabena Obeng Duedu, Julia Robinson

8 Apr 2022

Antibiotic-Resistant pathogenic bacterial isolates from patients attending the Outpatient Department of University of Cape Coast hospital, Ghana: A retrospective study between 2013-2015

PGPH-D-21-00765R1

Dear Dr Asare,

We are pleased to inform you that your manuscript 'Antibiotic-Resistant pathogenic bacterial isolates from patients attending the Outpatient Department of University of Cape Coast hospital, Ghana: A retrospective study between 2013-2015' has been provisionally accepted for publication in PLOS Global Public Health.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they'll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact globalpubhealth@plos.org.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Global Public Health.

Best regards,

Kwabena Obeng Duedu, PhD

Academic Editor

PLOS Global Public Health

***********************************************************

Reviewer Comments (if any, and for reference):

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

Reviewer #3: All comments have been addressed

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2. Does this manuscript meet PLOS Global Public Health’s publication criteria? Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe methodologically and ethically rigorous research with conclusions that are appropriately drawn based on the data presented.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available (please refer to the Data Availability Statement at the start of the manuscript PDF file)?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception. The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS Global Public Health does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: None

Reviewer #2: In an initial review, I made several comments and requested changes needed for the manuscript to be considered for publication. Most of the questions I raised have not been addressed. One important issue is the poor level of English of the manuscript that is still not addressed. Please see attached file for the content of my previous review.

Reviewer #3: Authors have done all necessary changes. So it can be published.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

Do you want your identity to be public for this peer review? If you choose “no”, your identity will remain anonymous but your review may still be made public.

For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Dr Enoch Aninagyei

Reviewer #2: Yes: Yakhya Dieye

Reviewer #3: No

**********

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Table. The patterns of isolation of resistant pathogens among males and females at the UCC hospital department.

    (XLS)

    Click here for additional data file. (33KB, xls)
    S2 Table. The antibiotics zone of inhibitions among the resistant pathogens isolated from 2013 to 2015.

    (XLS)

    Click here for additional data file. (37.5KB, xls)
    Attachment

    Submitted filename: 2.12.2021 Response to reviewers_Microbiology.doc

    Click here for additional data file. (93KB, doc)

    Data Availability Statement

    The authors confirm that the data supporting the findings of this research are available within the article.

    Articles from PLOS Global Public Health are provided here courtesy of PLOS

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