Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda

Richard Mayanja; Adrian Muwonge; Dickson Aruhomukama; Fred Ashaba Katabazi; Mudarshiru Bbuye; Edgar Kigozi; Annettee Nakimuli; Musa Sekikubo; Christine Florence Najjuka; David Patrick Kateete

doi:10.1371/journal.pone.0286955

. 2023 Jun 8;18(6):e0286955. doi: 10.1371/journal.pone.0286955

Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda

Richard Mayanja ^1,^2,^‡, Adrian Muwonge ^3,^*,^#, Dickson Aruhomukama ^1,², Fred Ashaba Katabazi ², Mudarshiru Bbuye ², Edgar Kigozi ², Annettee Nakimuli ^4,^‡, Musa Sekikubo ^4,^‡, Christine Florence Najjuka ^1,^*,^#, David Patrick Kateete ^1,^2,^*,^#

Editor: Mabel Kamweli Aworh⁵

PMCID: PMC10249850 PMID: 37289837

Abstract

Introduction

Escherichia coli, Klebsiella pneumoniae and Enterobacter (EKE) are the leading cause of mortality and morbidity in neonates in Africa. The management of EKE infections remains challenging given the global emergence of carbapenem resistance in Gram-negative bacteria. This study aimed to investigate the source of EKE organisms for neonates in the maternity environment of a national referral hospital in Uganda, by examining the phenotypic and molecular characteristics of isolates from mothers, neonates, and maternity ward.

Methods

From August 2015 to August 2016, we conducted a cross-sectional study of pregnant women admitted for elective surgical delivery at Mulago hospital in Kampala, Uganda; we sampled (nose, armpit, groin) 137 pregnant women and their newborns (n = 137), as well as health workers (n = 67) and inanimate objects (n = 70 –beds, ventilator tubes, sinks, toilets, door-handles) in the maternity ward. Samples (swabs) were cultured for growth of EKE bacteria and isolates phenotypically/molecularly investigated for antibiotic sensitivity, as well as β-lactamase and carbapenemase activity. To infer relationships among the EKE isolates, spatial cluster analysis of phenotypic and genotypic susceptibility characteristics was done using the Ridom server.

Results

Gram-negative bacteria were isolated from 21 mothers (15%), 15 neonates (11%), 2 health workers (3%), and 13 inanimate objects (19%); a total of 131 Gram-negative isolates were identified of which 104 were EKE bacteria i.e., 23 (22%) E. coli, 50 (48%) K. pneumoniae, and 31 (30%) Enterobacter. Carbapenems were the most effective antibiotics as 89% (93/104) of the isolates were susceptible to meropenem; however, multidrug resistance was prevalent i.e., 61% (63/104). Furthermore, carbapenemase production and carbapenemase gene prevalence were low; 10% (10/104) and 6% (6/104), respectively. Extended spectrum β-lactamase (ESBL) production occurred in 37 (36%) isolates though 61 (59%) carried ESBL-encoding genes, mainly bla_CTX-M (93%, 57/61) implying that bla_CTX-M is the ideal gene for tracking ESBL-mediated resistance at Mulago. Additionally, spatial cluster analysis revealed isolates from mothers, new-borns, health workers, and environment with similar phenotypic/genotypic characteristics, suggesting transmission of multidrug-resistant EKE to new-borns.

Conclusion

Our study shows evidence of transmission of drug resistant EKE bacteria in the maternity ward of Mulago hospital, and the dynamics in the ward are more likely to be responsible for transmission but not individual mother characteristics. The high prevalence of drug resistance genes highlights the need for more effective infection prevention/control measures and antimicrobial stewardship programs to reduce spread of drug-resistant bacteria in the hospital, and improve patient outcomes.

Introduction

The World Health Organization (WHO) estimates that 5 million neonatal deaths occur annually, disproportionately affecting populations in the developing countries. Septicaemia is among the leading causes of morbidity and mortality in neonates and infants in the developing countries [1]. In addition to causing common skin and urinary tract infections, members of the Enterobacteriaceae family, especially Escherichia coli, Klebsiella pneumoniae, and Enterobacter species (spp.) (herein EKE bacteria) are reported to be the leading cause of septicaemia in Africa [2]. Clinicians increasingly recognise septicaemia as a life-threatening condition due to organ failure resulting from host deregulations and cellular metabolic breakdown [3]. Therefore, immediate clinical management is needed, which is dominated by the use of β-lactam class of antibiotics, especially the extended-spectrum β-lactam agents like the third-generation cephalosporins (e.g., ceftriaxone, ceftazidime). However, Gram-negative bacteria, especially members of the Enterobacteriaceae family, have increasingly become resistant to third-generation cephalosporins [4]. This has made infections they cause increasingly difficult to manage. Phenotypically, drug resistant E. coli, K. pneumoniae, and Enterobacter spp. produce β-lactamases that block the action of antibiotics; genotypically, these bacteria harbour extended spectrum β-lactamase (ESBL) encoding genetic elements like bla_CTX-M, bla_TEM, and bla_SHV, as well as AmpC encoding genes like DHA, CMY, and CIT. ESBL-mediated resistance is prevalent among the Enterobacteriaceae in African settings, for example in Uganda and Tanzania, where management using cephalosporins has been reported [5]. Moreover, ESBL-producing Enterobacteriaceae carry additional genetic elements like bla_VIM, bla_IMP, bla_KPC, bla_OXA-48, and bla_NDM, which encode carbapenemases i.e., VIM (veronica integrin Metallo-beta-lactamases), IMP (imipenemase), KPC (Klebsiella pneumoniae carbapenemase), OXA-48 (oxacillinase-48), and NDM-1 (New Delhi Metallo-beta-lactamase-1), respectively [6]. The carbapenemases hydrolyse almost all β-lactam antibiotics [7] and enable resistance to carbapenems, a group of highly effective antibiotics [8].

Antibiotic resistant bacteria become ubiquitous when susceptible sub-populations that do not carry resistance genes are exposed to antibiotics that kill susceptible bacteria, thus selecting for resistant populations [9]. Furthermore, it is widely accepted that a history of visiting a hospital is a risk factor for acquisition of ESBL-producing Gram-negative bacteria, which are known for colonizing hospital surfaces, health workers, and pregnant women accessing prenatal hospital services [10,11]. This inherently makes neonates/infants an extremely high-risk group. Over the last 20 years, Mulago National Referral Hospital in Kampala, Uganda, has registered a considerable increase in neonatal morbidity and mortality predominantly caused by Gram-negative bacteria [12]. Moreover, studies conducted in Uganda have shown that ESBL-producing isolates are highly resistant to third-generation cephalosporins, specifically ceftazidime and cefotaxime [3], and that ESBL production occurs at variable levels at the hospital [5,12]. This, coupled with their ability to persist in hospital environments, makes ESBL-producing bacteria a significant health risk to neonates [13].

DNA amplification techniques combined with conventional phenotypic characterization of antibiotic resistance allows not only to cost-effectively ascribe phenotypic resistance to responsible genes, but also supports source-tracking at the hospital and community level in a resource-limited setting [14–16]. In this study, we examined the dispersal of ESBL-producing EKE bacteria in the maternity ward at Mulago hospital using phenotypic and genotypic characteristics of the isolates, and identified the potential source of drug resistant bacteria for neonates. This is critical for understanding the clinical and sanitary points of control, hence contributing to a reduction in hospital-acquired antimicrobial resistance.

Methods

Study setting

The study was conducted at Mulago hospital in Kawempe division, 3 km from Kampala city centre. Mulago serves as both the national referral hospital for Uganda and a teaching hospital for Makerere University; it is the largest public hospital in the country with 1,600 beds and a 1:40 doctor-to-patient ratio. It receives about 100 pregnant women daily, delivering up to 60 babies by ~50 midwives. Nearly half of these babies are born by Caesarean section [17]. According to hospital records there were 31,201 babies born in 2010, 33,331 in 2011, 33,231 in 2012 and 31,400 in 2013; in 2014, 30,000 babies were delivered at the hospital, which is about 68% of all the babies born in Uganda, giving it a claim to the top position of the busiest labour wards in the world [17].

Study design, participants and eligibility criteria

The study design was cross-sectional, centred around the routine maternity activities at the hospital. The study analysed Enterobacteriaceae isolates cultured from samples (swabs) collected in a parallel study that looked at community methicillin resistant Staphylococcus aureus (MRSA) carriage and nosocomial MRSA acquisition among pregnant women in the maternity ward during August 2015 and August 2016, Fig 1. With consent, 137 pregnant women (purposive sampling) admitted to the hospital for elective surgical delivery (Caesarean section) were recruited, and subsequently were the babies delivered by the women. Samples (nasal, armpit and groin swabs) were collected from the pregnant women at admission, delivery and discharge from the hospital, Fig 1; additionally, samples were collected from neonates following surgical delivery. In case a mother or baby developed sepsis, swabs were collected from wounds or the vagina or baby’s cord to investigate the cause of sepsis. Also, we collected 137 environment samples–from health workers (n = 67, hereafter animate samples) who were handling the mothers/babies, as well as beds, ventilator tubes, sinks, toilets, and door-handles (n = 70, hereafter inanimate samples) in the labour ward. Overall, approximately 820 swab samples were processed and investigated for growth of K. pneumoniae, E. coli and Enterobacter spp.

The laboratory procedures were carried out in the Clinical Microbiology and Molecular Biology Laboratories of the College of Health Sciences, Makerere University. The Clinical Microbiology Laboratory participates in the College of American Pathologists’ bacteriology external quality assurance scheme (CAP no. 7225593). In the laboratory, samples were inoculated on nonselective media (blood agar) and incubated overnight at 37°C in ambient air; among the isolates obtained, a significant number (n = 167) with features suggestive of Enterobacteriaceae were identified and stored in 20% brain heart infusion (BHI)-glycerol at -20°C. These are the isolates of interest that we retrieved and investigated; isolates were recovered by sub-culturing on blood agar at 37°C in ambient air for 18–24 hours, and sub-culturing on MacConkey agar at 37°C in ambient air for 12 hours. Identification to species level was based on phenotypic characteristics i.e., Gram staining and biochemical tests i.e., oxidase test, triple sugar iron agar (TSIA), indole, citrate utilization and urease production tests [18].

Antibiotic sensitivity testing

Antibiotic sensitivity testing was done with the Kirby-Bauer disc diffusion test [6] using sensitivity discs–ceftriaxone (CRO, 30 μg), cefotaxime (CTX, 30 μg), cefepime (FEP, 30 μg), ceftazidime (CAZ, 30 μg), cefoxitin (FOX, 30 μg), cefoxitin/cloxacillin (FOX/CLOX, 30 μg / 200 μg/ml), meropenem (MEM, 10 μg), meropenem/ethylenediaminetetraacetic acid (MEM/EDTA, 30 μg/100 μg/ml), ciprofloxacin (CIP, 5 μg), gentamicin (CN, 10 μg), chloramphenicol (C, 30 μg), tetracycline (TE, 30 μg), trimethoprim/sulfamethoxazole (SXT, 1.25/23.75μg), amoxicillin-clavulanate (AMC, 30 μg), aztreonam (ATM, 30 μg), and piperacillin-tazobactum (TPZ, 110 μg). Briefly, an inoculum was prepared from a pure culture plate of a test isolate grown overnight. This was done by touching with a sterile loop the top of 3-to-5 colonies with similar appearance, suspending in normal saline and adjusting turbidity to 0.5 McFarland (approximately 1.5 x 10⁸ colony forming units [CFU]). Adjusting the density of the test suspension to that of the standard was done by adding more bacterial suspension or sterile normal saline. A sterile cotton swab was dipped into the bacterial suspension, and excess liquid removed by rotating the swab several times with firm pressure on the inner wall of the tube above the fluid level. Using the swab, a Mueller Hinton Agar (MHA) plate was streaked to form a bacterial lawn. To obtain uniform growth, the plate was streaked with the swab in one direction, rotated at 60 degrees and streaked again in another direction. The rotation was repeated three times then the swab passed round the edge of the agar surface as it was drawn across the plate. The plate was allowed to airdry for about 3–5 minutes before adding the antibiotic disc. Using a sterile pair of forceps, the antibiotic disc was added to the media plate and gently pressed on the agar to ensure it was attached. MHA plates with antibiotic discs were incubated at 37°C overnight in ambient air, after which zones of inhibition (in mm) were measured using a divider and ruler, and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2015) [19].

ESBL screening

Isolates with inhibition zone diameters suggestive of ESBL production i.e., ceftriaxone (CRO) = 23 mm, cefotaxime (CTX) = 26 mm, aztreonam (ATM) = 21 mm, and ceftazidime (CAZ) = 21 mm [20] were screened for ESBL production using the double disc synergy test and the modified double disc synergy test (MDDST), in which cefepime (FEP) replaced ceftriaxone [21]. An amoxicillin-clavulanate disc (20/10 μg) along with four cephalosporins discs i.e., cefotaxime, ceftriaxone, ceftazidime, and cefepime, were used. A lawn culture of the test isolate was made on an MHA plate with an amoxicillin-clavulanate disc placed in the centre of the plate. Then, cefotaxime, ceftriaxone, ceftazidime, and cefepime discs were placed 20 mm centre-to-centre to the amoxicillin-clavulanate disc and incubated overnight at 37°C. Any distortion or increase in the zone of clearance towards the amoxicillin-clavulanate disc was considered positive for ESBL production; K. pneumoniae strain 700603 and E. coli strain 25922 were used as the positive and negative controls, respectively.

Screening for AmpC enzymes

Isolates with a cefoxitin inhibition zone diameter of ≤17 mm were screened for AmpC enzyme production using cefoxitin disc (30 μg) and cefoxitin (30 μg) + cloxacillin (200 μg) discs on MHA plates incubated overnight at 37°C. The inhibition zone diameter around the cefoxitin + cloxacillin disc was compared to that of cefoxitin without cloxacillin for confirmation of AmpC β-lactamase production. An inhibition zone diameter difference of ≥4 mm was interpreted as positive for AmpC production. Cloxacillin was used as the inhibitor for AmpC enzyme activity, while E. coli strain ATCC25922 was used as the negative control [6].

Screening for carbapenemases

Isolates with a meropenem (10 μg) inhibition zone diameter of ≤23mm were screened for carbapenemase production using the modified Hodge’s test (MHT) [22]. A 1:10 dilution of the indicator/susceptible organism (E. coli ATCC 25922) was adjusted to turbidity equivalent to 0.5 McFarland in normal saline, streaked on MHA plate and air-dried for 5–10 minutes, and a meropenem disc (10 μg) placed in the centre of the plate. Test isolates were streaked outward from the disc to the edge of the plate (20–25 mm in length) using a sterile swab. The same procedure was carried out for the positive control (K. pneumoniae ATCC^® BAA-1705™) and the negative control (K. pneumoniae ATCC^® BAA-1706™). Plates were incubated in ambient air for 16–20 hours at 37°C and results interpreted according to the CLSI (2015) guidelines. Briefly, β-lactamase production was verified based on distortion of the inhibition zone; a positive result had enhanced growth around the positive control streak at the intersection of the zone of inhibition (i.e., formation of a clover-leaf indentation of indicator strain growing along the streak of the test organism within the antimicrobial diffusion diameter); on the other hand, a negative result had no growth of the indicator strain along the streak of the test isolate within the disc diffusion zone.

To screen for metallo-beta-lactamase production, an overnight culture of a test isolate equivalent to 0.5 McFarland was inoculated on MHA plates using a sterile swab. After 5–10 minutes of drying, two meropenem discs (10 μg) were placed on the surface of the agar 15 mm apart, centre-to-centre. Ten microliters of 0.5 M EDTA was added to one of the meropenem discs and incubated at 37°C overnight. An increase in the zone of inhibition by ≥5 mm around the EDTA potentiated disc was interpreted as positive for metallo-β-lactamase production.

Detection of antibiotic resistance genetic elements

Isolates screened for ESBL and carbapenemase activity were molecularly investigated for ESBL- and carbapenemase gene carriage. We used conventional PCR to detect bla_CTX-M, bla_TEM, and bla_SHV genes which confer bacterial resistance to β-lactam agents except carbapenems and cephamycin [8]. We also used PCR to detect carbapenemase encoding genes bla_VIM, blaIMP, and bla_NDM. Except for the bla_CTX-M gene variants where we used inhouse primers, we used previously published primers and conditions for the PCRs [6,23], S1 Table. PCR amplicons were analysed by gel electrophoresis on a 1.5% agarose gel stained with ethidium bromide and viewing DNA bands in a UV trans-illuminator. Isolates that were previously confirmed to be positive or negative for the genes being investigated were used as positive and negative controls, respectively. Furthermore, PCR-amplicons were sequenced to confirm the resistance genes through BLAST searches at the National Centre for Biotechnology Information (NCBI) https://blast.ncbi.nlm.nih.gov/Blast.cgi

Data analysis

Disc diffusion using the Kirby Bauer method was interpreted according to CLSI guidelines [19]. Microsoft Excel 2016 and SPSS version 16.0 were used for data entry and statistical analyses. Differences in proportions and means were compared using chi square and the student t-test, respectively. A p-value of <0.05 was considered statistically significant. Cluster analysis of the phenotypic and genotypic characteristics of the isolates was done using Ridom GmBH, Münster, Germany. Here, the phylogenetic analysis module was used to cluster phenotypes or genotypes by similarity of profiles and then visualized using UPGMA phylogenetic tree.

Ethical considerations

Ethical approval was provided by the School of Biomedical Sciences Research and Ethics Committee at Makerere University (SBS-REC 434); a waiver of consent to use archived samples was provided by the SBS-REC. Authors did not have access to information that could identify individual participants during or after data collection.

Results and discussion

Participants’ demographics and bacterial isolates

In this study, purposive sampling was used to enrol 137 pregnant women who underwent Caesarean surgical delivery (C-section) and their natal babies (n = 137 –there were no multiple pregnancies), and screened for contamination with E. coli, K. pneumoniae and Enterobacter spp. Key demographics of the mothers are summarized in Table 1. Overall, Gram-negative bacteria were isolated from 21 mothers, 15 babies (neonates), 2 health workers, and 13 inanimate objects, Table 2. A total of 131 Gram-negative isolates were identified, of which 104 isolates were of our interest i.e., E. coli, K. pneumoniae and Enterobacter spp.; K. pneumoniae was the most prevalent species (38%, 50/131) followed by Enterobacter spp. (24%, 31/131), Table 2. Other Gram-negative bacteria were identified but not discussed further–these include Citrobacter spp., Pseudomonas spp., and Acinetobacter spp., Table 3.

Table 1. Demographics of pregnant women (n = 137) who underwent Caesarean surgical delivery and screened for isolation of E. coli, K. pneumoniae and Enterobacter spp.

Variable	Categorization	n (%)
Highest education level attained	Primary	30 (22)
	Secondary or Vocational	65 (47)
	Advanced level	25 (18)
	Tertiary institute^*	9 (7)
	Bachelor’s degree	8 (6)
Sample site (and time point)	Groin (on admission)	23 (17)
	Groin (on discharge)	58 (42)
	Armpit (on admission)	0
	Armpit (on discharge)	32 (23)
	Nose (on admission)	24 (18)

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* Tertiary but not university.

Table 2. E. coli, K. pneumoniae and Enterobacter spp. isolated and investigated (n = 104).

Source	E. coli (%)	K. pneumoniae (%)	Enterobacter spp. (%)	Total^*
Mother, 21/137 (15%)	05 (4)	18 (13)	07 (5)	30
Baby, 15/137 (11%)	05 (4)	15 (11)	10 (7)	30
Environment (animate): 2/67 (3%)	10 (15)	06 (9)	02 (3)	18
Environment (inanimate): 13/70 (19%)	03 (1)	11 (16)	12 (17)	26
Total (%)	23 (22)	50 (48)	31 (30)	104

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*Some participants/objects grew multiple bacterial species (i.e., polymicrobial samples).

Table 3. Other Gram-negative bacteria identified, n = 131 (%).

Source	Citrobacter	Acinetobacter	Pseudomonas	K. oxytoca	Total
Mothers (n = 137)	7 (5)	5 (4)	2 (2)	0	14
Babies (n = 137)	2 (2)	6 (5)	1 (0.8)	1 (0.8)	10
Health workers (n = 67)	6 (5)	7 (5)	2 (2)	0	15
Environment (Inanimate) (n = 70)	2 (2)	0	0	0	2
Total	17 (13)	18 (14)	5 (4)	1 (0.8)	41

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Antibiotic susceptibility profiles

The highest drug sensitivity level was noted for carbapenem antibiotics whereby 97% (30/31) of Enterobacter spp., 89% (44/50) of K. pneumoniae, and 82% (19/23) of E. coli isolates were susceptible to meropenem, Table 4. Nevertheless, 58% (29/50) of the K. pneumoniae isolates, 70% (16/23) of E. coli, and 58% (18/31) of Enterobacter spp. were multidrug resistant; to determine multidrug resistance (MDR), resistance to β-lactams, aminoglycosides, trimethoprim-sulfamethoxazole, tetracyclines and fluoroquinolones was considered. Table 5 depicts the resistance combinations noted and virtually all patterns involved a non-β-lactam agent; the most common MDR pattern among the K. pneumoniae isolates was gentamicin+chloramphenicol+trimethoprim-sulfamethoxazole while for E. coli and Enterobacter it was ciprofloxacin+gentamicin+chloramphenicol+tetracycline+trimethoprim-sulfamethoxazole.

Table 4. Antibiotic susceptibility characteristics of the EKE bacterial isolates n, (%)^*.

Species	AMC	TPZ	CRO	CTX	CAZ	ATM	FEP	FOX	MEM	SXT	CIP	CN	C	TE
E. coli (n = 23)	16 (70)	14 (61)	8 (35)	8 (35)	4 (17)	10 (44)	4 (17)	12 (52)	19 (83)	10 (44)	5 (22)	10 (44)	14 (61)	9 (39)
K. pneumoniae (n = 50)	30 (60)	25 (50)	18 (36)	13 (26)	21 (38)	21 (42)	18 (36)	41 (82)	44 (88)	13 (26)	5 (10)	26 (52)	19 (38)	36 (72)
Enterobacter (n = 31)	10 (32)	18 (58)	8 (26)	13 (42)	10 (32)	13 (42)	14 (45)	2 (7)	30 (97)	14 (45)	20 (65)	11 (36)	15 (48)	20 (65)

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AMC, ampicillin-sulbactam; TPZ, piperacillin-tazobactam; CRO, ceftriaxone; CTX, cefotaxime; CAZ, ceftazidime; ATM, aztreonam; FEP, meropenem; FOX, cefoxitin; MEM, meropenem; SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin; CN, gentamicin; C, chloramphenicol; TE, tetracycline.

*Refers to percentage of drug susceptible isolates.

Table 5. Multiple resistance patterns to classes of antibiotics.

Combination	K. pneumoniae n = 50 (%)	E. coli n = 23 (%)	Enterobacter spp., n = 31 (%)
C+SXT	5 (10%)	1 (4%)	0
C+TE	1 (2%)	0	0
C+CN	0	0	2 (6%)
CN+SXT	0	0	1 (3%)
CIP+SXT	0	1 (4%)	0
CIP+TE	2 (4%)	0	0
TE+SXT	1 (2%)	0	1 (3%)
CN+C+SXT	9 (18%)	0	0
CN+TE+SXT	0	1 (4%)	1 (3%)
CN+C+SXT	0	0	3 (10%)
CIP+C+SXT	2 (4%)	0	0
CIP+TE+SXT	1 (2%)	4 (17%)	0
CIP+CN+SXT	0	1 (4%)	0
C+TE+SXT	1 (2%)	1 (4%)	0
CIP+CN+TE+SXT	0	0	1 (3%)
CIP+CN+C+SXT	0	0	1 (3%)
CIP+C+TE+SXT	0	1 (4%)	0
CN+C+TE+SXT	5 (10%)	1 (4%)	2 (6%)
CIP+CN+C+TE+SXT	2 (4%)	5 (22%)	6 (19%)

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SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin; CN, gentamicin; C, chloramphenicol; TE, tetracycline.

Characterisation of beta-lactamases and carbapenemases

ESBL, AmpC and carbapenemase activity was detected in all the three species, Table 6; ESBL activity was highest in K. pneumoniae (50%) while AmpC activity was highest in Enterobacter spp. (45%). However, carbapenemase activity was comparatively low, Table 6.

Table 6. Prevalence of beta-lactamases and carbapenemases.

Species	ESBLs alone (%)	AmpC alone (%)	ESBL+AmpC (%)	Carbapenemases (%)
E. coli (n = 23)	9 (39)	5 (22)	6 (26)	3 (13)
K. pneumoniae (n = 50)	25 (50)	6 (12)	3 (6)	6 (12)
Enterobacter spp. (n = 31)	3 (10)	14 (45)	12 (39)	1 (3)

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Characteristics of genetic determinants of antibiotic resistance

Tables 7 and S2 depict the frequency and distribution of the antibiotic resistance genetic elements associated with resistance to β-lactams and carbapenems. Overall, ESBL and carbapenemase encoding genes were detected in 59% (61/104) of the isolates and the former were more prevalent particularly the bla_CTXM-U/15 gene. While bla_CTX-M, bla_TEM and bla_SHV occurred in isolates regardless of the presence of phenotypic ESBL-activity, bla_VIM, bla_NDM and bla_IMP occurred in only carbapenemase-producing isolates. Furthermore, bla_CTX-M-15 was the only bla_CTX-M gene found in E. coli and Enterobacter spp. while it occurred in 23 of the 29 ESBL gene positive K. pneumoniae isolates, implying that the six K. pneumoniae isolates with the universal bla_CTX-M-U gene carried other bla_CTX-M types. Overall, these data show that bla_CTX-M-15 is a predominant ESBL encoding gene in this setting. Furthermore, carriage of multiple resistance genetic elements was frequent in K. pneumoniae and Enterobacter spp., especially ESBL encoding genes and the most common pattern was bla_CTXM-U/15 + bla_SHV, implying that the bla_TEM and bla_SHV genes in this setting are co-transmitted with bla_CTXM-U/15 in that carriage of bla_TEM alone or bla_SHV alone wasn’t seen. On the other hand, the carbapenemase encoding genes were less prevalent and occurred in only six isolates, S2 Table. Note, while the carbapenemase gene prevalence is low in this study, four of the six carbapenemase gene positive isolates (i.e., bla_VIM+, bla_IMP+, bla_NDM+) co-carried the genes, and almost all were ESBL gene positive, S2 Table.

Table 7. Summary of the antibiotic resistance genes among PCR-positive isolates.

Species	bla _CTXM-U	bla _CTXM-15	bla _TEM	bla _SHV	bla _VIM	bla _IMP	bla _NDM
E. coli n = 23 (%)	12 (52)	12 (52)	7 (30)	3 (13)	02 (9)	02 (9)	02 (9)
Klebsiella n = 50 (%)	29 (58)	23 (46)	10 (20)	23 (46)	02 (4)	01 (2)	0
Enterobacter n = 31 (%)	16 (52)	16 (52)	09 (29)	3 (10)	01 (3)	0	0
Total	57	51	26	29	05	03	02

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Inferring transmission from clustering of drug resistance phenotypes and genotypes

For an insight into the source/transmission of MDR E. coli, K. pneumoniae and Enterobacter spp. in the maternity ward, spatial cluster analysis of phenotypic and genotypic susceptibility characteristics was performed and the analyses presented as dendrograms for inferring relationships, Figs 2 & 3. Based on phenotypic susceptibility characteristics, seven clusters comprising two or more isolates from mothers, their babies, health workers (animate), and/or environment (inanimate) were noted, Fig 2. Additionally, isolates from mothers with susceptibility characteristics similar to isolates from babies that were not their own were noted. Still, based on molecular susceptibility characteristics, eight clusters comprising two to eight isolates from mothers, their babies, health workers (animate), and/or environment (inanimate) were noted, Fig 3. Overall, these data allude to occurrence of epidemiological links for the clustered isolates hence, transmission in the maternity ward of MDR E. coli, K. pneumoniae and Enterobacter spp. from mothers to new-borns.

Fig 2 — Depicts seven clusters comprising drug resistant isolates of E. *coli*, K. *pneumoniae* and *Enterobacter* spp. with similar antibiotic susceptibility profiles hence, potential transmission of MDR bacteria from mothers/environment to new-borns. Clusters of isolates with similar susceptibility characteristics are denoted with an asterisk (*).

Fig 3 — Depicts isolates of E. *coli*, K. *pneumoniae* and *Enterobacter* spp. with similar molecular susceptibility profiles hence, potential transmission of MDR bacteria from mothers to new-borns in the maternity ward. Asterisks (*) denote isolates from mothers, babies and/or environment with similar genotypic characteristics.

Overall, this study depicts a high recovery of K. pneumoniae, Enterobacter and E. coli with phenotypic and genotypic characteristics of multi-resistance in the maternity ward of Mulago hospital. The clustering of phenotypic and genotypic profiles by time and space suggests active transmission between mother and new-born babies, as well as health workers and their maternity ward environment. This calls into question the effectiveness of infection prevention and control strategies, given the isolation of these potential pathogens from healthcare equipment, ward environment and the patients. However, the fact that bacteria were also isolated from participants on admission into the hospital, there is an indication that some of the profiles could be acquired from the community before the mothers are admitted, suggesting a role of community as a contributor to the diversity of organisms observed in this study, Fig 4.

In addition, the high prevalence of K. pneumoniae, Enterobacter and E. coli in mothers and their natal babies depict the hygiene levels of items mothers use on hospital admission. Our findings are in line with studies in similar settings for example, Kayange et al (2010) who looked at the predictors of positive blood culture and deaths among neonates with suspected neonatal sepsis in a tertiary hospital in Mwanza, Tanzania [1]. Also, in Kenya a study that investigated hospital acquired infections in a private pediatrics’ hospital found K. pneumoniae to be the most prevalent species followed by Pseudomonas aeruginosa and Enterobacter cloacae [24].

Moreover, the high MDR levels among isolates in this study suggests high selection pressure in the hospital [25], as well as overuse and inappropriate use of antibiotics [26]. Indeed, there was high carriage of antibiotic resistance encoding genes (especially ESBL genes) among isolates, which means organisms have acquired resistance genes and disseminated them to other organisms, for example, through plasmids that can carry various genes [27]. Among the ESBL-encoding genes, bla_CTX-M, bla_SHV and bla_TEM were detected at rates comparable to previous studies [28]. Though, we found bla_CTX-M to be the most frequent gene while previous studies in similar settings reported bla_TEM (48.7%) to be the most prevalent, followed by bla_CTX-M (7.6%) and bla_SHV (5.1%) [28]. We also found carriage of antibiotic resistant isolates by health workers to be comparable to that of studies done elsewhere [29–31]. Health workers can acquire bacterial contamination by direct contact with patients, body fluids secretions, or touching contaminated environmental surfaces within the hospital environment [32]. Just like in earlier studies in Uganda [6,23,33], the carbapenemase gene prevalence remains low at Mulago hospital; despite this, we identified carriage of more than one carbamenemase gene, suggesting enhanced drug resistance to carbapenems. Finally, spatial cluster analysis suggests transmission between animate and inanimate or a shared source of contamination for the maternity ward; this source could be items like health worker’s gloves, stethoscopes and other items which were not sampled in our study [34].

Limitations

The transmission investigation could have benefited from a more granular analytical method such as next generation sequencing to better track the source using single nucleotide polymorphisms. Due to resources this was not possible, nonetheless the findings provide clues that will be further examined as and when resources become available. Also, the data utilized in the study was gathered in the period between 2015 and 2016, and considering that a lot of transformations might have taken place since then, it may not accurately depict the present circumstances at the hospital. In light of this, we suggest carrying out a similar study that employs updated datasets for a contemporary overview of the drug resistance situation at Mulago.

Conclusions

Our findings suggest a potentially high exposure rate of mothers and their new-born babies to a variety of MDR Enterobacteriaceae strains when admitted for elective surgical delivery. The potential sources of these strains were health workers, maternity ward environment as well as introductions from the community by mothers. The findings suggest inadequacies in infection control practices on the maternity ward. Given the high prevalence of ESBLs on the ward, we recommend regular review of the infection control protocols in the maternity ward, and more studies should be conducted to look at other organisms and sample more items used in the hospital especially those which are shared by patients. Molecular techniques with a high discriminatory power such as DNA sequencing and/or Pulsed Field Gel electrophoresis should be considered in future studies.

Supporting information

S1 Checklist. STROBE statement—Checklist of items that should be included in reports of observational studies.

(DOCX)

Click here for additional data file.^{(36KB, docx)}

S1 Table. Primer sequences used to PCR-amplify ESBL-encoding and carbapenemase-encoding genes.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S2 Table. Frequency and distribution of antibiotic resistance genetic elements among PCR-positive isolates.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

Acknowledgments

Special thanks to staff at the Departments of Immunology and Molecular Biology and Medical Microbiology, Makerere University College of Health Sciences for the support they provided during the time the research was conducted, as well as the research participants who agreed to be part of this study. Finally, special thanks to the Cambridge group that collected the isolates and made them available to us.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

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

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PLoS One. doi: 10.1371/journal.pone.0286955.r001

Decision Letter 0

Mabel Kamweli Aworh

3 Apr 2023

PONE-D-23-06934Source-tracking ESBL-producing bacteria at the maternity ward of Mulago Hospital, UgandaPLOS ONE

Dear Dr. Kateete,

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Reviewer #1: This is a very informative and technical work. Few inputs made; work is good for publishing.

I appreciate the fact that you numbered your lines making reviewing this work a lot easier, though some numbered lines are blank I would use your numbering as is. Thank you

1. Line 34, page 9; please correct cross-section study to cross-sectional study

2. Lines 50 - 52, page 10; please review this statement and clarify

3. Lines 114 – 115, page 12; do you have access to more recent birth records? If yes, please use that unless you have a good reason for using these from 9 years ago.

4. Line 132, page 13; please do not abbreviate the word approximately

5. Line 137, page 14; please change carried out from to “carried out in “

6. Line 240, page 18; please use chi 2 or chi square

7. Lines 242 - 243, page 18; please change was to were

8. Include your keywords to the written manuscript

9. The omission of gloves used by health workers as part of where samples should be taken was a huge oversight in this work nevertheless much work was put into this study and it is very technical and highly informative. The work itself has a lot of papers embedded

10. Justice was done to the title

Reviewer #2: Thank you for this important study that characterizes the sources of ESBL which is crucial for infection control and prevention at a tertiary hospital ward in Uganda .

The Abstract is well written, however, the authors should consider adding the policy or practice implication to the abstract. Additionally, line 24 reads cross-section instead of cross-sectional.

Major strengths of this study include the well-written methodology and the presentation of the result findings in tables that are clear and easy to understand. A major weakness of this study is the time that the data was collected which was more than five years ago. however, the study still provides important insights.

The Introduction gives a good background and a good justification for the study.

Methods:

Line 108: Consider removing "isolates" as there is no description of isolates in this section.

Line 125: The authors should consider consistency in describing the sample collection sites e.g. nasal is referred to nares and anterior nares in different sections; armpit is referred to as axillar (line 264; also note that the correct word is axilla)

Line 127: The authors should consider changing "as well" to other phrases such as Also, Additionally etc.

Line 132: approx. should be be written in full or changed to another term. Same as for Line 161.

Line 173: Consider adding a 'comma' after "ruler"

Results and Discussion:

Line 251: The authors should consider writing "discussion" as a sentence case

Line 261: further should be in the past tense.

Line 328: The authors should consider changing "as well" to other phrases such as Also, Additionally etc.

Line 360: Kayange et al should be written as Kayange et al., and the year of the article should be included.

Limitations:

Line 387: The authors should consider writing the none the less as one word.

Also consider acknowledging that the data was collected in 2015-2016 and any implication if any.

Conclusions: Line 396: Consider reworking "carrying out more studies" to "more studies should be conducted". Also consider rephrasing "urgent infection control protocol" as this data for this study was collected between 2015-2016, and a lot of things might have changed.

Overall, this was a well conducted and reported study with necessary ethical approvals obtained.

Reviewer #3: The manuscript was well laid out with a good method section; However, authors need to explain how they arrived at a sample of 137 women? is it a purposive sampling technique? Religion of participants were listed in Table 1, and it is unclear how that relates to having ESBL producing bacteria. Consider dropping religion. Other minor edits as commented in the attached file

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Reviewer #1: Yes: Folashade Onatola Bamidele

Reviewer #2: No

Reviewer #3: Yes: Augustine Olajide Dada

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PLoS One. 2023 Jun 8;18(6):e0286955. doi: 10.1371/journal.pone.0286955.r002

Author response to Decision Letter 0

9 May 2023

May 9, 2023

The Academic Editor, PLOS ONE,

Dear Dr. Mabel Kamweli Aworh,

RE: Point-by-point response to reviewers – Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda (PONE-D-23-06934)

We thank you for considering our manuscript, and for forwarding to peer-reviewers for critique. We are indebted for the comments/suggestions and have carefully considered all of them – including comments in the attachments on email, and improved the manuscript accordingly.

Kindly find our point-by-point response to each concern, below. We outline how the comments have been addressed and point to specific line numbers and pages where to find the changes.

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1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

Response:

The revised manuscript meets PLOS ONE’s style requirements.

2. Thank you for stating the following in the Acknowledgments Section of your manuscript: "Special thanks to staff at the Departments of Immunology and Molecular Biology and Medical Microbiology, Makerere University College of Health Sciences for the support they provided during the time the research was conducted, as well as the research participants who agreed to be part of this study. Finally, special thanks to the Cambridge group that collected the isolates and made them available to us. AM is a Chancellor’s Fellow at the Roslin Institute and his time was paid core funding and as a Future leader fellow funded by BBSRC (BB/P007767/1) and Wellcome Trust ISSF3 (IS3-R1.09 19/20).”

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows: "The author(s) received no specific funding for this work."

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

Response:

We have removed funding-related text from the revised manuscript.

Kindly update our Funding Statement as;

"The author(s) received no specific funding for this work. AM is a Chancellor’s Fellow at the Roslin Institute and his time was paid core funding and as a Future leader fellow funded by BBSRC (BB/P007767/1) and Wellcome Trust ISSF3 (IS3-R1.09 19/20)."

As advised, we have included the above statement in the cover letter for you to change the online submission on our behalf.

Response:

All the cited references have been cross checked online and to the best of our knowledge, none has been retracted. We realize that some journals might be no longer in production, for example SpringerPlus that published article No. 23 was discontinued however, the article itself is not retracted – it appears online and is searchable.

RESPONSE TO REVIEWERS' COMMENTS:

REVIEWER #1

This is a very informative and technical work. Few inputs made; work is good for publishing. I appreciate the fact that you numbered your lines making reviewing this work a lot easier, though some numbered lines are blank I would use your numbering as is.

Response: We are delighted to know that you found our manuscript informative and worth publishing in PLOS ONE. We have dully addressed all the concerns you raised; please find our response below;

Comment 1: Line 34, page 9; please correct cross-section study to cross-sectional study

Response: Thank you – this has been corrected, see line 34.

Comment 2: Lines 50 - 52, page 10; please review this statement and clarify.

Response: Apologies for our not being clear here. All we intended to say is that we employed a data analysis procedure referred to as ‘spatial cluster analysis’, in which we investigated the phenotypic and genotypic drug susceptibility characteristics of the isolates and inferred relationships among them. Accordingly, the statement has been rephrased for clarity – it now reads as “Additionally, cluster analysis revealed that isolates from mothers, newborns, health workers, and environment have similar phenotypic and genotypic characteristics, suggesting transmission of multidrug-resistant EKE bacteria to newborns” (lines 51-53). For a better perspective on this, we also reviewed the methods section to read ‘To infer relationships among the EKE isolates, spatial cluster analysis of the phenotypic and genotypic antibiotic susceptibility profiles was done using the Ridom server.’, see lines 40-41.

Comment 3: Lines 114 – 115, page 12; do you have access to more recent birth records? If yes, please use that unless you have a good reason for using these from 9 years ago.

Response: Apologies – we are unable to obtain the most up-to-date birth records for Mulago hospital at this moment. The information we shared is based on data made available to the public at that point in time.

Comment 4: Line 132, page 13; please do not abbreviate the word approximately

Response: The word approximately is now written in full in the revised manuscript.

Comment 5: Line 137, page 14; please change carried out from to “carried out in “

Response: As suggested, the phrase ‘Carried out from’ has been changed to ‘carried out in’ (line 143)

Comment 6: Line 240, page 18; please use chi 2 or chi square

Response: chi2 has been changed to chi square, line 245

Comment 7: Lines 242 - 243, page 18; please change was to were

Response: Here we are referring to ‘cluster analysis’ hence we have maintained ‘was’ as it is singular; however, we have rephrased for clarity, see lines 246-248.

Comment 8: Include your keywords to the written manuscript

Response: Keywords have been included, line 60

Comment 9: The omission of gloves used by health workers as part of where samples should be taken was a huge oversight in this work nevertheless much work was put into this study and it is very technical and highly informative. The work itself has a lot of papers embedded

Response: We concur with the reviewer's assessment that the failure to include information about the gloves used by healthcare workers in relation to where samples were taken was a significant oversight in our research. As we drew our data from a parent study that did not account for this information, it was not available for our analysis. We appreciated your understanding of this limitation, which we indeed acknowledged and will consider in further studies.

Comment 10: Justice was done to the title

Response: We thank you for your appreciation.

REVIEWER #2

Comment (Abstract / Introduction):

• Thank you for this important study that characterizes the sources of ESBL which is crucial for infection control and prevention at a tertiary hospital ward in Uganda.

• The Abstract is well written, however, the authors should consider adding the policy or practice implication to the abstract. Additionally, line 24 reads cross-section instead of cross-sectional.

• Major strengths of this study include the well-written methodology and the presentation of the result findings in tables that are clear and easy to understand. A major weakness of this study is the time that the data was collected which was more than five years ago. however, the study still provides important insights.

• The Introduction gives a good background and a good justification for the study.

Response:

We thank you for your comments and for your appreciation of the important insights our manuscript provides. As you have advised, the policy / practice implication has been provided to the revised abstract, see lines 56 to 59. Also, in agreement with REVIEWER 1, we have changed ‘cross-section’ to ‘cross-sectional’, line 34.

Comment (Methods):

• Line 108: Consider removing "isolates" as there is no description of isolates in this section.

• Line 125: The authors should consider consistency in describing the sample collection sites e.g. nasal is referred to nares and anterior nares in different sections; armpit is referred to as axillar (line 264; also note that the correct word is axilla)

• Line 127: The authors should consider changing "as well" to other phrases such as Also, Additionally etc.

• Line 132: approx. should be be written in full or changed to another term. Same as for Line 161.

• Line 173: Consider adding a 'comma' after "ruler"

Response:

• The word “isolates” has been removed as suggested, line 113

• In this revision, “nare” has been replaced with “nose” and “axilla” has been replaced with “armpit”, see Table 1. Also, this reminded us of the informed consent procedure and documents, in which study participants (mothers) were told that a swab will be picked from their nose and armpit.

• The phrase “as well” has been replaced with “additionally”, line 132

• In the revised version, the word “approx.” has been written in full throughout the text

• A coma has been added after ‘ruler’, see line 179.

Comment (Results and Discussion):

• Line 251: The authors should consider writing "discussion" as a sentence case

• Line 261: further should be in the past tense.

• Line 328: The authors should consider changing "as well" to other phrases such as Also, Additionally etc.

• Line 360: Kayange et al should be written as Kayange et al., and the year of the article should be included.

Response:

• As suggested, discussion has been written as sentence case, line 256.

• As suggested, the sentence now reads as “Other Gram-negative bacteria were identified but not discussed further”, line 265-267.

• The “As well” phrase has been replaced with “Additionally”, see line 334.

• The suggested change has been made, see line 369.

Comment (Limitations):

• Line 387: The authors should consider writing the none the less as one word.

• Also consider acknowledging that the data was collected in 2015-2016 and any implication if any.

Response:

• The phrase “none the less” has been rewritten as one word i.e., “nonetheless”, line 396.

• We thank you; the acknowledgement of data collection in 2015-2016 and implication thereof, have been included, see lines 397-402

Comment (Conclusions):

• Line 396: Consider reworking "carrying out more studies" to "more studies should be conducted". Also consider rephrasing "urgent infection control protocol" as this data for this study was collected between 2015-2016, and a lot of things might have changed.

• Overall, this was a well conducted and reported study with necessary ethical approvals obtained.

Response:

• The phrase "carrying out more studies" has been changed to "more studies should be conducted", line 410-411.

• Further, in light of your suggestion the phrase "urgent infection control protocol" has been revised and toned down, see lines 405-414.

REVIEWER #3

Comment:

• The manuscript was well laid out with a good method section; However, authors need to explain how they arrived at a sample of 137 women? is it a purposive sampling technique?

• Religion of participants were listed in Table 1, and it is unclear how that relates to having ESBL producing bacteria. Consider dropping religion.

Response:

• Thank you for mentioning that our manuscript is well-laid out. As you have hinted, for sample size we used purposive sampling to select the 137 pregnant women scheduled for caesarian surgical delivery. So, in the revised manuscript we have added a statement to this effect, see lines 129 & 258.

• As advised, data on religion has been removed, see Table 1.

Attachment

Submitted filename: Response-to-Reviewers.docx

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PLoS One. doi: 10.1371/journal.pone.0286955.r003

Decision Letter 1

Mabel Kamweli Aworh

26 May 2023

Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda

PONE-D-23-06934R1

Dear Dr. KATEETE,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Academic Editor

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Reviewer #3: Yes

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Reviewer #3: Yes

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6. Review Comments to the Author

Reviewer #1: All previous recommendations by reviewers have been corrected accordingly hence good for publishing. It is a good work with good information for health facilities, health care workers and IPC enthusiasts . The experiments were made clearly with ability for replication.

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PLoS One. doi: 10.1371/journal.pone.0286955.r004

Acceptance letter

Mabel Kamweli Aworh

1 Jun 2023

PONE-D-23-06934R1

Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda

Dear Dr. Kateete:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Dr. Mabel Kamweli Aworh

Academic Editor

PLOS ONE

Associated Data

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

Supplementary Materials

S1 Checklist. STROBE statement—Checklist of items that should be included in reports of observational studies.

(DOCX)

Click here for additional data file.^{(36KB, docx)}

S1 Table. Primer sequences used to PCR-amplify ESBL-encoding and carbapenemase-encoding genes.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

S2 Table. Frequency and distribution of antibiotic resistance genetic elements among PCR-positive isolates.

(DOCX)

Click here for additional data file.^{(16.8KB, docx)}

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Source-tracking ESBL-producing bacteria at the maternity ward of Mulago hospital, Uganda

Richard Mayanja

Adrian Muwonge

Dickson Aruhomukama

Fred Ashaba Katabazi

Mudarshiru Bbuye

Edgar Kigozi

Annettee Nakimuli

Musa Sekikubo

Christine Florence Najjuka

David Patrick Kateete

Roles

Abstract

Introduction

Methods

Results

Conclusion

Introduction

Methods

Study setting

Study design, participants and eligibility criteria

Fig 1. Study schematic depicting participants and sampling timelines.

Antibiotic sensitivity testing

ESBL screening

Screening for AmpC enzymes

Screening for carbapenemases

Detection of antibiotic resistance genetic elements

Data analysis

Ethical considerations

Results and discussion

Participants’ demographics and bacterial isolates

Table 1. Demographics of pregnant women (n = 137) who underwent Caesarean surgical delivery and screened for isolation of E. coli, K. pneumoniae and Enterobacter spp.

Table 2. E. coli, K. pneumoniae and Enterobacter spp. isolated and investigated (n = 104).

Table 3. Other Gram-negative bacteria identified, n = 131 (%).

Antibiotic susceptibility profiles

Table 4. Antibiotic susceptibility characteristics of the EKE bacterial isolates n, (%)*.

Table 5. Multiple resistance patterns to classes of antibiotics.

Characterisation of beta-lactamases and carbapenemases

Table 6. Prevalence of beta-lactamases and carbapenemases.

Characteristics of genetic determinants of antibiotic resistance

Table 7. Summary of the antibiotic resistance genes among PCR-positive isolates.

Inferring transmission from clustering of drug resistance phenotypes and genotypes

Fig 2. Cluster analysis of phenotypic susceptibility characteristics.

Fig 3. Cluster analysis of genotypic susceptibility characteristics.

Fig 4. Hypothetical sources of MDR K. pneumoniae, E. coli and Enterobacter spp. in the maternity ward of Mulago hospital.

Limitations

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Mabel Kamweli Aworh

Roles

Author response to Decision Letter 0

Decision Letter 1

Mabel Kamweli Aworh

Roles

Acceptance letter

Mabel Kamweli Aworh

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases

Table 4. Antibiotic susceptibility characteristics of the EKE bacterial isolates n, (%)^*.