Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda.

Staphylococci are a key component of the human microbiota, and they mainly colonize the skin and anterior nares. However, they can cause infection in hospitalized patients and healthy individuals in the community. Although majority of the Staphylococcus aureus strains are coagulase-positive, some do not produce coagulase, and the isolation of coagulase-positive non-S. aureus isolates in humans is increasingly being reported. Therefore, sound knowledge of the species and characteristics of staphylococci in a given setting is important, especially isolates from children and immunocompromised individuals. The spectrum of Staphylococcus species colonizing children in Uganda is poorly understood; here, we aimed to determine the species and characteristics of staphylococci isolated from children in Eastern Uganda. Seven hundred and sixty four healthy children less than 5 years residing in Iganga and Mayuge districts in Eastern Uganda were enrolled. A total of 513 staphylococci belonging to 13 species were isolated from 485 children (63.5%, 485/764), with S. aureus being the dominant species (37.6%, 193/513) followed by S. epidermidis (25.5%, 131/513), S. haemolyticus (2.3%, 12/513), S. hominis (0.8%, 4/513) and S. haemolyticus/lugdunensis (0.58%, 3/513). Twenty four (4.95%, 24/485) children were co-colonized by two or more Staphylococcus species. With the exception of penicillin, antimicrobial resistance (AMR) rates were low; all isolates were susceptible to vancomycin, teicoplanin, linezolid and daptomycin. The prevalence of methicillin resistance was 23.8% (122/513) and it was highest in S. haemolyticus (66.7%, 8/12) followed by S. aureus (28.5%, 55/193) and S. epidermidis (23.7%, 31/131). The prevalence of multidrug resistance was 20.3% (104/513), and 59% (72/122) of methicillin resistant staphylococci were multidrug resistant. Four methicillin susceptible S. aureus isolates and a methicillin resistant S. scuiri isolate were mupirocin resistant (high-level). The most frequent AMR genes were mecA, vanA, ant(4')-Ia, and aac(6')-Ie- aph(2'')-Ia, pointing to presence of AMR drivers in the community.

Introduction

Staphylococci are a key component of the human microbiota, and around 47 species have been identified [1, 2]. They mainly colonize the skin, anterior nares, the nasopharynx, and gut [2-4]. Staphylococci are generally nonpathogenic however, when there is breach of the body barriers (e.g. damage to the skin and mucous membranes) or when the immune system is weakened, they become opportunistic pathogens and cause a variety of mild to severe community- and hospital-acquired infections [2-4].

One of the most useful characteristics used to differentiate staphylococci is their ability to produce coagulase. In this regard, staphylococcal isolates are broadly classified into two groups–the coagulase-positive staphylococci (CoPS) comprising of mainly Staphylococcus aureus, which is more pathogenic, and the coagulase-negative staphylococci (CoNS) that are less pathogenic but include majority of the species [2]. While majority of S. aureus strains are overwhelmingly coagulase-positive, some are atypical in that they do not produce coagulase [5-11]. Among the CoNS, the medically relevant species include S. epidermidis, S. haemolyticus, S. hominis and S. saprophyticus [1, 2]. Interestingly, one species, Staphylococcus schleiferi, includes both CoNS and CoPS subspecies i.e. S. schleiferi subsp. schleiferi and S. schleiferi subsp. coagulans, respectively [2]. Furthermore, there are five additional CoPS species i.e. S. delphini, S. hyicus, S. intermedius, S. intrae and S. pseudintermedius, which are typically animal-associated [2] but they have also been isolated from human samples. Although these non-S. aureus CoPS species are characteristically animal-associated [2], they are likely to be prevalent in human specimens in the developing countries [5] where rural, agrarian populations predominate.

Staphylococcal colonization is a known risk factor for staphylococcal disease [12]. Globally, there is an increase in prevalence of nosocomial infections caused by CoNS and atypical S. aureus, and this is attributed to the increase in use of procedure-related devices in human medicine [2]. CoNS and atypical S. aureus infections are difficult to treat as they are more likely to be methicillin-resistant and multidrug-resistant; moreover, CoNS species like S. lugdenensis and S. saprophyticus are associated with severe infections i.e. infective endocarditis and urinary tract infects, respectively [2]. It is therefore important to unambiguously identify staphylococci to species-level, particularly isolates from vulnerable groups such as children and immunocompromised individuals. Additionally, an understanding of the dynamics of staphylococcal colonization in a given setting, especially methicillin resistant CoNS (MRCoNS), is critical as they are considered to be the reservoir of antibiotic resistance genes for the more pathogenic species (S. aureus) [3, 13]. Several studies have reported transfer of the staphylococcal cassette chromosome mec (SCCmec), a transposon that bears the molecular determinant of methicillin resistance (mecA gene), between S. epidermidis and S. aureus [14]. While the role of CoNS as reservoirs of the SCCmec element is well-established, the spectrum of species potentially involved in SCCmec exchange is not well documented, particularly in the developing countries. Here we aimed to determine the species and characteristics of staphylococci isolated from children in Eastern Uganda. As virulence and antimicrobial resistance (AMR) tend to coexist in staphylococci [15, 16], we also determined the frequency of genes associated with these phenotypes among community-associated staphylococci in Eastern Uganda.

Materials and methods

Ethics statement

This study was approved by the Institutional Review Boards of the Schools of Medicine, Public Health, and Biomedical Sciences at Makerere University College of Health Sciences (REF 2011–183; SBS194), and by the Uganda National Council for Science and Technology (Ref HS 1080) [17-20]. Written informed consent was obtained from the parents/guardians of the children, and the consent procedure included consent for storage of samples for further studies. Since the study was conducted on stored isolates, the Research Ethics Committees waived the need for re-consenting of the participants.

Study setting, samples and isolates

This cross-sectional study was nested in previous studies/projects that investigated the pneumococcal nasopharyngeal colonization and integrated community case management of malaria and pneumonia in children less than 5 years of age at the Iganga/Mayuge Health and Demographic Surveillance Site (IMHDSS) in rural Eastern Uganda [20, 21]. The study population was described before [20-22]; briefly, 764 healthy children less 5 years at the IMHDSS were enrolled and sampled. Following consent, a study nurse collected two nasopharyngeal samples (swabs) from each child. Swabs were transported in small batches to the Clinical Microbiology laboratory in Kampala for culturing. Samples were processed for isolation of staphylococci by following standard microbiology procedures described previously [5, 20] (and summarized below).

Species identification and antimicrobial susceptibility testing

Following overnight culturing of samples on non-selective medium (blood agar plates), Gram-positive and catalase-positive isolates were sub-cultured again on solid Brain Heart Infusion (BHI) medium and confirmed to species level by using the Phoenix 100 Identification (ID) / Antimicrobial Susceptibility Testing (AST) Automated Microbiology System from the Becton, Dickinson and Company (Franklin Lakes, New Jersey), as described previously [23, 24]. Morphologically distinct colonies with characteristic appearance of staphylococci were selected for automated identification. Antibiotic susceptibility testing by minimum inhibitory concentrations (MICs) to a panel of 16 antibiotics (cefoxitin, penicillin, ampicillin, tetracycline, trimethoprim/sulfamethoxazole, erythromycin, chloramphenicol, gentamicin, ciprofloxacin, rifampicin, clindamycin, mupirocin [high-level], teicoplanin, linezolid, vancomycin and daptomycin) was determined with the Phoenix Automated Microbiology System as previously described [23, 24] [25]. Multidrug resistance (MDR) was defined as isolates resistant to three or more classes of antimicrobials. To validate automated species’-level identification by the Phoenix ID/AST Expert System, we randomly selected 24 isolates representing presumptively identified isolates of S. aureus, S. epidermidis and S. haemolyticus, and molecularly re-identified them by polymerase chain reaction (PCR) as described previously [26]. To determine the SCCmec types among the methicillin resistant staphylococci (MRS), SCCmec typing was done and interpreted as described previously [17, 22, 27]. For quality control, S. aureus ATCC™ 29213 and Enterococcus faecalis ATCC™ 29212 were included in the Phoenix ID/AST panels and molecular assays.

Detection of virulence and AMR genes

PCR was performed to detect the virulence and AMR encoding genes, which tend to co-exist in invasive strains [15, 16]. Crude genomic DNA used as template in the PCRs was extracted by boiling for 3–5 minutes, freshly cultured cells re-suspended in 200 μl of Tris-EDTA (TE) buffer containing 80 U/ml lysostaphin. The Panton Valentine Leukocidin encoding genes (LukS-PV and LukF-PV), as well as the ica/D, bhp, atlE, hla, hld, hlg, tsst and sea genes, were detected by PCR as described previously [19, 28]. Similarly, presence of the genes encoding the aminoglycoside-modifying enzymes (AMEs) i.e. aac(6')-Ie-aph(2'')-Ia (bifunctional aminoglycoside-6-N-acetyltransferase/2''-O-phosphoryltransferase), aph(3')-IIIa (aminoglycoside-3'-O-phosphoryltransferase III) and ant(4')-Ia (aminoglycoside-4'-O-nucleotidyltransferase I), as well as the vanA/vanB1 genes that encode vancomycin resistance variants, was determined by PCR as described previously [19, 28, 29].

Results

Nasopharyngeal colonization rates and Staphylococcus species identified

Staphylococci were isolated from 485 of the 764 children (63.5%, 485/764); there was no growth of staphylococci or other bacteria in samples from 279 children (36.5%, 279/764). The 485 samples with Staphylococcus growth yielded a total of 513 isolates belonging to 13 species, Table 1. S. aureus was the most prevalent species at 37.6% (193/513) followed by S. epidermidis (25.5%, 131/513), S. haemolyticus (2.3%, 12/513), S. hominis (0.8%, 4/513) and S. haemolyticus/lugdunensis (0.58%, 3/513), Table 1 and S1 Table. However, the overall prevalence of CoNS including the 156 CoNS isolates that could not be identified to species level was 42% (320/764), higher than that of S. aureus, Table 1. As expected the sole isolate of S. intermedius was coagulase-positive but nuc-PCR negative, and it was identified to species level by the Phoenix ID/AST expert system.

Table 1

Staphylococcus species recovered from the children (n = 764).

Species	Sub-totala n, (%)	MDR n, (%)	Methicillin resistant n, (%)	mecA n, (%)
S. aureus	193/513 (38)	58/193 (30)	55/193 (29)	55/193 (29)
S. epidermidis	131/513 (26)	14/131 (11)	31/131 (24)	31/131 (24)
S. haemolyticus	12/513 (2)	12/12 (100)	8/12 (67)	8/12 (67)
S. hominis	4/513 (1)	1/4 (25)	0	0
S. haemolyticus/ lugdunensis	3/513 (1)	0	2/3 (67)	2/3 (67)
S. pasteuri	3/513 (1)	3/3 (100)	3/3 (100)	3/3 (100)
S. lentus	2/513 (0.4)	0	1	1
S. capitis	2/513 (0.4)	1	0	0
S. xylosus	2/513 (0.4)	2	2	2
S. kloosii	2/513 (0.4)	2	2	2
S. intermedius	1/513 (0.2)	0	0	0
S. caprae	1/513 (0.2)	1	1	1
S. sciuri	1/513 (0.2)	1	1	1
Other CoNSb	156/513 (30)	9/156 (6)	16/156 (10)	16/156 (10)
Total	513	104/513 (20)	122/513 (24)	122/513 (24)
Co-colonizedc	24 children (5)	17 children (71)	13 children (54)	13 children (54)

aOf 513 staphylococcal isolates recovered.

Refers to other coagulase negative staphylococci that could not be identified to species level

cOf 485 children in whom staphylococci were isolated

Overall, nasopharyngeal colonization rates by the dominant species was as follows: 25.3% (193/764) S. aureus, 17.1% (131/764) S. epidermidis, and 1.6% (12/764) S. haemolyticus. A total of 24 (4.95%, 24/485) children were co-colonized by two or three Staphylococcus species and the commonest co-colonizing species were S. aureus and S. epidermidis. Of note, 66.7% (16/24) of the co-colonized children harbored a multidrug resistant Staphylococcus while 58.3% (14/24) had a MRS; only three of the co-colonized children harbored distinct MRS species, Table 2.

Table 2

Co-colonization of the children by various Staphylococcus species.

Child #	Species	Bacteriological evaluation
		MRS	MDR	mecA	vanA	AMEs	LukS-PV/LukF-PV	tst1	ica	hla	hld	bhp
H0005	S. aureus	No	No	-	-	-	-	-	-	+	+	-
	S. epidermidis	No	No	-	+	-	-	-	-	+	+	+
H0007	S. aureus	No	Yes	-	-	-	-	-	-	-	+	-
	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
H0008	S. aureus	Yes	Yes	+	-	-	-	-	-	+	+	-
	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
H0231	S. aureus	No	Yes	-	-	-	-	-	-	-	-	-
	S. epidermidis	No	No	-	+	-	-	-	-	+	+	-
H0354	S. aureus	No	No	-	-	-	-	-	-	-	-	-
	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
H0184	S. aureus	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	Yes	Yes	+	-	-	-	-	-	+	+	+
H0403	S. aureus	Yes	Yes	+	-	-	-	-	-	-	-	-
	S. xylosus	Yes	Yes	+	-	-	-	-	-	-	-	-
H0473	S. aureus	No	Yes	-	-	-	-	+	-	+	+	-
	S. kloosii	Yes	Yes	+	-	-	-	-	-	+	+	-
H0023	S. aureus	Yes	No	+	-	-	-	-	-	+	+	-
	CoNS	Yes	No	+	-	-	-	-	-	+	+	-
H0271	S. aureus	No	Yes	-	-	-	-	-	-	-	-	-
	S. epidermidis	Yes	Yes	+	-	-	-	-	-	-	+	-
	S. haemolyticus / lugdunensis	Yes	No	+	-	-	-	-	-	-	+	-
H0029	S. aureus	No	No	-	-	-	-	-	-	-	-	-
	S. epidermidis	Yes	No	+	-	+	-	-	-	+	+	-
	CoNS	No	No	-	-	-	-	-	-	-	-	-
H0034	S. aureus	Yes	Yes	+	-	-	-	-	-	+	+	-
	S. epidermidis	Yes	Yes	+	-	-	-	-	-	+	-	-
	S. hominis	No	No	-	-	-	-	-	-	-	-	-
H0057	S. aureus	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	Yes	Yes	-	-	-	-	-	-	+	+	+
	S. pasteuri	Yes	Yes	-	-	-	-	-	-	+	+	+
H0108	S. epidermidis	Yes	Yes	+	-	+	-	-	-	-	+	-
	S. haemolyticus	Yes	Yes	+	-	+	-	-	-	-	+	-
H0020	S. epidermidis	Yes	Yes	+	-	-	-	-	-	-	-	-
	S. haemolyticus	Yes	Yes	+	-	-	-	-	-	-	-	-
H0251	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	No	Yes	-	-	-	-	-	-	-	-	-
H0328	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	No	Yes	-	-	-	-	-	-	-	-	-
H0332	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	No	Yes	-	-	-	-	-	-	-	-	-
H0185	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus	No	Yes	-	-	-	-	-	-	-	-	-
H0160	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus / lugdunensis	No	No	-	-	-	-	-	-	-	-	-
H0067	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. haemolyticus / lugdunensis	Yes	No	+	-	-	-	-	-	-	-	-
H0150	S. epidermidis	No	No	-	-	-	-	-	-	-	-	+
	S. intermedius	No	No	-	-	-	-	-	-	-	-	-
H0227	S. epidermidis	No	No	-	-	-	-	-	-	-	-	-
	S. sciuri	Yes	Yes	+	-	-	-	-	-	-	-	-
H0341	S. epidermidis	Yes	Yes	+	-	-	-	-	-	-	+	-
	S. capitis	No	No	+	-	-	-	-	-	-	+	-

Children co-colonized with a MRS are indicated in boldface font. +, PCR positive (gene detected); -, PCR negative (gene not detected).

Antimicrobial susceptibility patterns

The overall prevalence of methicillin resistance was high at 23.8% (122/513); per species methicillin resistance was highest in S. haemolyticus (66.7%, 8/12) followed by S. aureus (28.5%, 55/193) and S. epidermidis (23.7%, 31/131). The overall prevalence of MRCoNS was 20.9% (67/320). All MRS isolates were mecA positive however, they were fully susceptible to anti-MRSA agents–teicoplanin, linezolid, vancomycin and daptomycin. Also, MRS isolates were highly susceptible to mupirocin, rifampicin, chloramphenicol, and clindamycin, S1 and S2 Tables. Overall, with the exception of penicillin/methicillin, antimicrobial resistance rates were generally low especially to chloramphenicol, rifampicin, clindamycin, vancomycin, teicoplanin, linezolid and daptomycin, Table 3 and S1 Table. Five isolates with high-level mupirocin resistance (HLMupr) were identified, four of which were methicillin susceptible S. aureus (MSSA) while one was a methicillin resistant S. scuiri (MRSS). Rifampicin resistance was detected in only one isolate, a methicillin resistant S. pastueri (MRSP). Compared to S. epidermidis, S. aureus isolates were more resistant to trimethoprim-sulfamethoxazole (SXT) (73/193, 38% vs. 13/131, 10%), erythromycin (37/193, 19% vs. 11/131, 8%), gentamicin (31/193, 16% vs. 4/131, 3%), ciprofloxacin (22/193, 11% vs. 4/131, 3%), cefoxitin (55/193, 29% vs. 31/131, 2%) and tetracycline (42/193, 22% vs. 22/131, 17%). As well, resistance among S. aureus isolates to cefoxitin (31/131, 24%), tetracycline (42/193, 22%), SXT (73/193, 38%), erythromycin (37/193, 19%), and ciprofloxacin (22/193, 11%) was less common than S. haemolyticus isolates (cefoxitin 8/12, 67% resistant; tetracycline 7/12, 58% resistant; SXT 12/12, 100% resistant; erythromycin 11/12, 92% resistant; ciprofloxacin 4/12, resistant), Table 3 and S1 Table. While we found few isolates of S. pastueri, S. xylosus and S. kloosii, they were all MRS and multidrug resistant, Table 1.

Table 3

Summary of antimicrobial susceptibility profiles of staphylococci from children in rural eastern Uganda (n = 513).

Species	No. isolates	Resistance to a panel of 15 antimicrobials n, (%)
		PEN	SXT	FOX	TET	ERY	GEN	CIP	CHL	MUP	CLI	RIF	TEI	LIN	VAN	DAP
S. aureus	193	174/193 (90)	73/193 (38)	55/193 (29)	42/193 (22)	37/193 (19)	31/193 (16)	22/193 (11)	6/193 (3)	4/193 (2)	3/193 (2)	0	0	0	0	0
S. epidermidis	131	124/131 (95)	13/131 (10)	31/131 (24)	22/131 (17)	11/131 (8)	4/131 (3)	4/131 (3)	0	0	1/131 (1)	0	0	0	0	0
S. haemolyticus	12	12/12 (100)	12/12 (100)	8/12 (67)	7/12 (58)	11/12 (92)	2/12 (17)	4/12 (33)	0	0	1/12 (8)	0	0	0	0	0
S. hominis	4	4	1	0	1	0	0	0	0	0	0	0	0	0	0	0
S. haemolyticus / lugdunensis	3	2	0	2	1	0	0	0	0	0	0	0	0	0	0	0
S. pasteuri	3	3	3	3	2	3	1	3	0	0	2	1	0	0	0	0
S. lentus	2	2	1	1	1	0	0	0	0	0	1	0	0	0	0	0
S. capitis	2	2	1	0	0	1	0	0	0	0	0	0	0	0	0	0
S. xylosus	2	2	2	2	2	2	0	2	0	0	2	0	0	0	0	0
S. kloosii	2	2	2	2	2	2	1	1	0	0	1	0	0	0	0	0
S. intermedius	1	1	0	0	0	0	1	0	0	0	0	0	0	0	0	0
S. caprae	1	1	1	1	1	1	0	0	0	0	0	0	0	0	0	0
S. sciuri	1	1	0	1	0	0	0	1	0	1	0	0	0	0	0	0
Other CoNS	156	134/156 (86)	16/156 (10)	16/156 (10)	19/156 (12)	11/156 (7)	3/156 (2)	3/156 (2)	0	0	0	0	0	0	0	0
Total	513	462/513 (90)	127/513 (25)	122/513 (24)	98/513 (19)	79/513 (15)	41/513 (8)	40/513 (8)	4/513 (1)	4/513 (1)	11/513 (2)	1/513 (0.2)	0	0	0	0

CoNS denotes other coagulase negative staphylococci that were not identified to species level in this study

FOX, cefoxitin; PEN, penicillin; TET, tetracycline; SXT, trimethoprim-sulfamethoxazole; ERY, erythromycin; CHL, chloramphenicol; GEN, gentamicin; CIP, ciprofloxacin; RIF, rifampicin; CLI, clindamycin; MUP, Mupirocin High level; TEI, teicoplanin; LIN, linezolid; VAN, vancomycin; DAP, daptomycin

The overall prevalence of MDR was 20.3% (104/513), and 59% (72/122) of the MRS were multidrug resistant, S2 Table. All S. haemolyticus and S. pastueri isolates were multidrug resistant while 30.1% (58.1/513) and 10.8% (14/131) of S. aureus and S. epidermidis isolates respectively, were multidrug resistant, Table 1. The prevalence of MDR was also high among the methicillin resistant S. aureus (MRSA) and methicillin resistant S. epidermidis (MRSE) i.e. 64.2% (34/53) and 42.4% (14/33), respectively. As expected, the methicillin resistant isolates were generally resistant to the non-beta lactam antimicrobials i.e. tetracycline (53%), SXT (55%), erythromycin (42%), and ciprofloxacin (32%). Overall, only 14.4% of CoNS were multidrug resistant but the MDR phenotype was generally high among MRCoNS.

The most frequent SCCmec type among MRS was type I (32.8%, 40/122) while types II and III were detected in 8.2% (10/122) and 6.6% (8/122) of MRS, respectively. SCCmec types I+II+III combined occurred in 47.5% (58/122) of MRS isolates. On the other hand, SCCmec types IV and V were detected in 18% (22/122) and 3.3% (4/122) of MRS, respectively, while SCCmec types IV+V combined occurred in 21.3% (29/122) isolates. Among MRSA, SCCmec types I and IV were the most frequent at 38.2% (21/55) and 31% (17/55) respectively; seven isolates were not type-able (NT). Likewise among MR-CoNS, SCCmec type I was the most prevalent at 6% (19/320) followed by types II (2.2%, 7/320), III (2.2%, 7/320) and IV (2%, 6/320); 28 MRCoNS were not type-able.

Virulence and antimicrobial resistance genes

In this study, all isolates were investigated for carriage of AMR and virulence genes in addition to the mecA gene described above. The most frequent AMR genes in S. aureus isolates were vanA, ant(4')-Ia, and aac(6')-Ie- aph(2'')-Ia while the most frequent virulence genes were hld and hla, Table 4. Although phenotypic resistance to vancomycin was not detected, 20 isolates carried the vanA gene while 6 carried the vanB gene. Eleven (42%, 11/26) of the isolates with vanA/vanB also carried the mecA gene. Furthermore, 62 isolates carried the AMEs genes (any of aac(6')-Ie-aph(2'')-Ia, ant(4')-Ia and aph(3')-IIIa), of which five (8%) were phenotypically resistant to gentamicin (an aminoglycoside). Generally, the hld, hla, sea and LukS-PV / LukF-PV genes were more frequent in S. aureus compared to CoNS species, Table 4. Additionally, in S. aureus the bhp gene was more frequent in MRSA isolates compared to MSSA while in CoNS, the sea, sdrE, bhp, hla, and hld genes were more frequent in MRSCoNS, Table 4. Among the CoNS, the AMR genes were more frequent in S. epidermidis compared to other species. The hlg gene was detected in only four isolates while hlb and ica/D genes were not detected. Overall, the virulence and AMR genes were generally more frequent in S. aureus compared to CoNS species (for example, the LukS-PV / LukF-PV genes were only detected in S. aureus).

Table 4

Frequency of virulence and antimicrobial resistance genes in staphylococci.

Gene	Function	No. isolates positive (%)
		S. aureus			CoNS
		All S. aureus	MRSA	MSSA	All CoNS	MRS	MSS
Antimicrobial resistance genes
mecA	Methicillin resistance	55/193 (29)	55/55 (100)	0	67/320 (21)	67/67 (100)	0
vanA	Vancomycin resistance (high-level)	11/193 (6)	4/55 (7)	7/138 (5)	9/320 (3)	3/67 (5)	6/253 (2)
vanB	Vancomycin resistance (low-level)	5/193 (3)	5/55 (9)	2/138 (1)	1/320 (0.3)	1/67 (2)	0
aac(6')-Ie- aph(2'')-Ia	AME* (GEN-R, TOB-R, AMK-R, KAN-R)	9/193 (5)	2/55 (4)	7/138 (5)	11/320 (3)	8/67 (12)	3/253 (1)
ant(4')-Ia	AME* (GEN-S, TOB-R, AMK-R, KAN-R)	14/193 (7)	3/55 (6)	11/138 (8)	10/320 (3)	7/67 (10)	3/253 (1)
aph(3')-IIIa	AME* (GEN-S, TOB-S, AMK-R, KAN-R)	3/193 (2)	0	3/138 (2)	3/320 (1)	3/67 (5)	0
Virulence genes
LukS-PV / LukF-PV**	Bicomponent leukocidin; pore-forming toxin; kills leukocytes	23/193 (12)	10/55 (18)	13/138 (9)	0
sea	Staphylococcal enterotoxin A	18/193 (9)	3 (6)	15/138 (11)	15/320 (5)	12/67 (18)	3/253 (1)
tst1	Endothelial toxicity (direct & cytokine-mediated); super-antigen activity	4/193 (2)	3 (6)	1/138 (1)	2/320 (1)	2/67 (3)	0
sdrE	Adhesin	10/193 (5)	3 (6)	7/138 (5)	13/320 (4)	11/67 (16)	2/253 (1)
bhp	Cell wall associated biofilm protein	22/193 (11)	11 (20)	11/138 (8)	28/320 (9)	16/67 (24)	12/253 (5)
hla	Cytolytic pore-forming toxin	54/193 (28)	19 (35)	35/138 (25)	48/320 (15)	32/67 (48)	16/253 (6)
hld	Cytolytic toxin; binds neutrophils & monocytes	77/193 (40)	24 (44)	53/138 (38)	85/320 (27)	53/67 (79)	32/253 (13)
Hlg	Bicomponent leukocidin; hemolysis	1/193 (1)	1 (2)	0	3/320 (1)	2/67 (3)	1/253 (0.4)

*AME(s) denotes aminoglycoside-modifying enzyme(s) AAC(6’)/APH (2''), ANT(4')-I and APH(3')-III encoded by the following genes: aac(6')-Ie- aph(2'')-Ia, ant(4')-Ia and aph(3')-IIIa, respectively; GEN, gentamicin; TOB, tobramycin; AMK, amikacin; KAN, kanamycin; S, susceptible, R, resistant.

**LukS-PV / LukF-PV, panton-valentine leukocidin; sea; tst1, toxic shock syndrome toxin-1; sdrE, Serine-aspartate repeat protein E; bhp, biofilm associated protein; hla, alpha-toxin; hlb, beta-toxin; hld, delta-toxin; hlg, gamma-toxin

Discussion

In this study, we used a rigorous approach to determine the species and characteristics of staphylococci colonizing children in Eastern Uganda. The Automated system we used i.e. the Phoenix AST/ID Expert System, is more accurate than the manual methods at identifying Gram-positive bacteria, as well as AMR testing [30]. However, in the developing countries, manual methods are the mainstay for identification of staphylococci, and in most cases the tube coagulase test is the confirmatory test for identification of S. aureus [5]. The tube coagulase test is considered to be more reliable at identifying S. aureus when a firm clot that doesn’t move on tilting the tube occurs; therefore, the subjectivity in interpreting the tube coagulase test probably leads to misidentification of CoNS as S. aureus [31]

In this study, a high staphylococcal nasopharyngeal colonization rate (63.5%, 485/764) was reported for healthy children in Eastern Uganda. However, our rate is comparable to the rates reported by other investigators e.g. ≥80% by Budri et al [32] and Faria et al [1]. Furthermore, the number of Staphylococcus species (i.e. 13) in this study is also comparable to the 9 species reported by Faria et al in Denmark [1], but less than the 19 species reported by Xu et al in a study of staphylococcal colonization in healthy individuals and the environment in London [14]. S. aureus was the most prevalent species followed by S. epidermidis, which contradicts reports of S. epidermidis as the most prevalent Staphylococcus species among staphylococcal isolates from humans [1, 14, 32]. However, as the overall prevalence of CoNS in this study (i.e. 42%, 320/764) was higher than that of S. aureus (37.6%, 193/513), the prevalence of S. epidermidis might be higher than what we have reported had we succeeded in identifying all the CoNS to species level. Still, several factors could have affected our recovery of S. epidermidis; for instance, unlike S. aureus, CoNS especially S. epidermidis mainly colonize the skin [33] yet we sampled the nasopharynx which is mainly colonized by S. aureus. Additionally, the enrichment methods employed in isolation of staphylococci generally favor recovery of S. aureus [32]. Interestingly, while majority of the CoNS species identified are human-associated (i.e. the S. epidermidis-like group–S. epidermidis, S. haemolyticus, S. capitis, S. hominis, etc. [2]), animal-associated CoNS species (i.e. S. caprae) and animal-associated CoPS species (i.e. S. intermedius) were also detected, which perhaps reflects a rural, agrarian population typical of Eastern Uganda. The detection of animal-associated staphylococci in this setting is a cause for concern as it may reflect (1) contamination of human samples by animal-associated strains, (2) occurrence of animal-associated CoPS in human samples, which could yield false-positive results on coagulase testing and overestimation of S. aureus/MRSA rates [30].

Around 5% of the children in this study were colonized by two or three Staphylococcus species, fewer than reports by investigators from Europe (i.e. 7.5% to 30%) [1, 3, 32]. In agreement with previous reports [1, 3, 32], the commonest co-colonizing species were S. aureus and S. epidermidis. Only one of the co-colonized children harbored S. aureus and S. haemolyticus/lugdunensis, which is not surprising as S. lugdnensis produces lugdunin, an antibacterial agent that reduces the probability of S. aureus colonization [3, 34]. Further, 66.7% (16/24) of the co-colonized children harbored a multidrug resistant Staphylococcus while 58.3% (14/24) harbored a MRS. However, only three of the co-colonized children harbored different species of MRS, and this is in agreement with the previously reported negative correlation of co-colonization by distinct MRS species [1, 3, 32].

Antimicrobial resistance rates were generally low across species. However, in sharp contrast to reports from the Nordic countries [35], penicillin resistance and MRS nasopharyngeal colonization rates were quiet high, which is consistent with global reports of increasing MRS prevalence in the community [36]. The detection of SCCmec types I+II+III combined and SCCmec types IV+V combined suggests coexistence of hospital-associated and community-associated MRS that seems to result from dissemination of hospital-associated strains into the community [36]. Further, there were five isolates with high-level mupirocin resistance (HLMupr), four of which were MSSA while one was a methicillin resistant S. scuiri (MRSS). This is consistent with increasing levels of mupirocin resistance staphylococci, especially in MRCoNS [3] [37], which is worrying as mupirocin is the drug of choice for eradication of Staphylococcus colonization. The occurrence of HLMupr in Africa before widespread use of mupirocin for MRSA decolonization requires continuous monitoring.

With the exception of mecA-positive isolates, majority of the isolates harboring AMR genes i.e. vanA/vanB and AMEs (any of aac(6')-Ie-aph(2'')-Ia, ant(4')-Ia and aph(3')-IIIa) were phenotypically susceptible to drug(s) targeted by the gene products. For example, phenotypic resistance to vancomycin was not detected; however, we found 26 isolates with the vanA/vanB genes, and 42% (11/26) of them carried mecA. vanA and vanB genes encode high- and low-level resistance to vancomycin, respectively. vanA type of resistance is widely spread in enterococci and it is transferrable between enterococci and staphylococci [29, 38, 39]. While phenotypic resistance to vancomycin among enterococci and staphylococci is rare in Uganda [24, 40], vanA/vanB PCR-positive vancomycin-susceptible isolates of enterococci and staphylococci have been reported in Uganda [28, 40]. While it is puzzling, vanA-positive-vancomycin-susceptible enterococci do occur, and they have also been reported from the developed countries [39, 41, 42]. These isolates have been found to lack key genes e.g. vanR and vanS, which are required for activation of transcription of the vancomycin resistance genes. Additionally, the vanA-positive-vancomycin-susceptible isolates can possess insertion sequences e.g. the ISL3-family element that silence transcription of the vanA operon [39, 41, 42]. The fact that horizontally transferrable silenced-vanA successfully reverted into resistance during vancomycin treatment [39], genotypic testing of invasive vancomycin-susceptible isolates has been recommended [33]. Relatedly, while phenotypic resistance to aminoglycosides correlates well with the detection of AMEs genes [43, 44], in this study, few isolates with AMEs genes (i.e. 8%, 5/62) were phenotypically resistant to gentamicin (an aminoglycoside). However, gentamicin-susceptible isolates positive for AMEs genes have been reported before [37, 38]. Overall, the carriage of AMR genes in absence of significant antibiotic selective pressure points to presence of AMR drivers in the community that should be investigated [32, 36] with robust approaches like whole genome sequencing [42] to better understand the mechanisms underlying altered AMR susceptibility in our setting.

In this study, the virulence and AMR genes were generally more frequent among S. aureus isolates compared to CoNS species. For example, the LukS-PV / LukF-PV genes were detected only in S. aureus but not CoNS. The hlg gene was detected in only four S. aureus isolates, while hlb and ica/D were not detected at all. The low frequency of virulence genes in CoNS is consistent with reports that CoNS generally possess a smaller repertoire of virulence genes compared to S. aureus [2], and that human-associated S. aureus strains do not express beta toxins (hlb) [33]. Furthermore, the bhp gene was prevalent in S. aureus and it was significantly associated with MRSA. Interestingly, bhp encodes a protein homologue of Bap (biofilm associated protein) and it is generally absent in human-associated S. aureus strains. However, the Bap homologue termed bhp, is present in S. epidermidis [2]. Investigators in the developed countries found an association between presence of bhp and aacA [aac(6)-aph(2)] genes with the “not cured” clinical outcome among patients who underwent surgery and got infected by staphylococci [45].

The fact that staphylococci are opportunistic pathogens, identification of virulence/invasive strains is critically important in guiding diagnosis and treatment of staphylococcal infections [46]. To this end, the detection of staphylococcal virulence factors, especially genes involved in biofilm formation, is frequently cited as a means through which we can rapidly identify invasive strains [19, 28, 46]. Compared to previously characterized staphylococcal isolates in Uganda [19, 28], we noted that the virulence and AMR genes are significantly more prevalent in S. aureus and generally in hospital isolates. In fact, the detection of icaA/D and hlg genes have been reported in hospital isolates but not community isolates [19, 28], and current study conforms to this notion.

The polysaccharide intercellular adhesin/poly-N-acetylglucosamine (PIA/PNAG) is associated with biofilm formation in staphylococci especially S. epidermidis. PIA/PNAG is encoded by the ica gene cluster, which comprises the icaA, icaD, icaB and icaR genes. Deletion of these genes is associated with biofilm absence [2, 46]. Biofilm production and presence of the ica operon correlate with disease causing clinical isolates and in mouse models, the PIA/PNAG-negative mutants were significantly less pathogenic [2, 28]. The bhp/Bap genes are also associated with the biofilm phenotype and may be useful as markers in distinguishing between pathogenic strains and normal flora [46] [2]. Overall, the implication of detecting these genes in flora of children at the IMHDSS, albeit at lower frequencies relative to hospital-associated isolates, is that (a) strains bearing these genes could be potentially virulent and, (b) the genes could be useful as markers for screening potentially invasive strains.

Conclusions

The staphylococcal nasopharyngeal colonization rate in children in Eastern Uganda is high but comparable to rates from other countries. Also, the Staphylococcus species’ distribution in the children mirrors what has been described from other settings but with S. aureus as the dominant species. Approximately 5% of the children were colonized by two or three Staphylococcus species but co-colonization by distinct MRS species is rare. Although the AMR rates were low, nasopharyngeal colonization by MRS is quiet high. The detection of high-level mupirocin resistance requires further investigation. Furthermore, while majority of CoNS and CoPS were human-associated, the detection of animal-associated CoNS and CoPS implies that individuals at the IMHDSS could be infected by zoonotic staphylococci. Importantly, with the occurrence of animal-associated CoPS species in human samples, tube-coagulase testing probably overestimates S. aureus/MRSA rates in this setting. Therefore, species-level identification of staphylococci with robust approaches is advised, especially clinically relevant isolates. The carriage of AMR genes in community-associated isolates, especially the vanA-positive-vancomycin-susceptible bacteria, points to existence of AMR drivers in the community that should be investigated for a better understanding of genetic determinants of altered antibiotic susceptibility. Overall, virulence and AMR genes, especially ica and bhp/Bap, were significantly more prevalent in S. aureus, could be useful as markers for screening potentially invasive strains.

General characteristics of community-associated staphylococci isolated from children less than 5 years in rural Eastern Uganda.

(XLS)

Click here for additional data file.

Details of drug resistance profiles of methicillin resistant staphylococci from children (n = 122).

(DOCX)

Click here for additional data file.

8 Nov 2019

PONE-D-19-21368

Species and susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda

PLOS ONE

Dear Dr. Kateete,

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Specific comments:

1. The rational in abstract should be strengthened.

2. Line 44 and 47, please use Staphylococcus aureus on line 44 and use S. aureus on line 47.

3. Line 56, please spell out SCCmec since this is the first time it was mentioned.

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PLOS ONE

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Reviewer #1: In this work, Kateete and colleagues determined the species and the antibiotic susceptibility of Staphylococci isolated from 764 healthy children under 5 years of age residing at the Iganga/Mayuge Health & Demographic Surveillance Site (IMHDSS) in Eastern Uganda. They isolated 513 staphylococci strains belonging to 13 different species. The most prevalent was Staphyloccoccus aureus, followed by S. epidermidis (25,5%), S. heamolyticus (2,3%), S. hominis (0,8%), and S. haemolyticus/lugduniensis (0.58%). The staphylococcal carriage rates from healthy children in rural Eastern Uganda were comparable to those from other countries showing a high level of virulence and AMR genes particularly in S. aureus strains.

Although restricted to a specific area in Africa, the identification of virulent staphylococci may offer some relevant information to the local clinical community, especially with the widespread occurrence of multidrug-resistant organisms and the increasing number of immunocompromised persons in the population. Continuous epidemiological surveillance would also contribute to reducing the Staphylococcal infection burden, hospital stay, and infection morbidity.

The manuscript is a well written, organized, and thorough presentation of the topic. I enjoyed reading the article and found it both relevant in its possible implications for the prevention and the development of targeted treatments and preventive strategies against invasive strains in children.

There are some minor typos throughout the text, which can be easily corrected.

Reviewer #2: This study reports the prevalence of staphylococcal nasopharyngeal colonisation amongst healthy children in Eastern Uganda. 764 children were included, and the authors identified the species of each isolate, their antimicrobial susceptibility and also tested for some virulence and resistance genes.

The sampling appears to have been at a single time point, so “colonisation” should be used rather than “carriage” – which suggests sampling longitudinally. Secondly, only the nasal pharynx was sampled – so “colonisation” should be “nasopharyngeal colonisation” throughout.

Acronyms should be defined when they are first used. MDR defined line 170 – but used prior. A definition of MDR needs to be provided in the methods.

Proportions have been provided to 1, 2, and 0 decimal places. This needs to be consistent, and I would recommend using whole numbers only. P values should be reported as <0.05 (and or <0.001). There is no section to explain what tests were used to derive P values – nor what hypotheses were actually being tested. In some instances I don’t think the hypothesis testing is useful – e.g. it would be more useful to the reader to list the % of each species resistant to each antibiotic than provide the low P values, which is less informative. The statistical analysis needs to be described in the methods.

How did the detection of resistance genes relate to the AST?

The genes listed should be consistent – LukSF or PVL is used (LukSF is more correct).

From line 213 the isolates are compared to another set of isolates. There is no introduction as to where these isolates came from, apart from saying that they were from a hospital. Unless a lot of further information is provided about the provenance of these isolates and why comparison with the study isolates is important or interesting then this section should be removed.

In line 126 the genus name does not require italics.

Line 143 “MRS staphylococcus” is not correct

MRS not defined until line 157.

Line 276/277 virulence genes and resistance genes confused

Line 280 does not with the preceding sentence.

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Reviewer #2: Yes: S R Ritchie

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26 Nov 2019

November 26, 2019

The Academic Editor, PLOS ONE

Dear Dr. Baochuan Lin, Ph.D.

RE: Submission of revised “PONE-D-19-21368”: Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda.

We thank you for handling our manuscript through the peer-review process, and finding it suitable for publication in PLOS ONE.

We carefully considered and fully effected all the insightful comments from the peer-reviewers, as well as your specific concerns. We removed the awkward sentences in the manuscript. To the best of our ability, we have copy-edited the manuscript for language usage, spelling, and grammar. We hope that you will find our manuscript suitable for publication in your esteemed journal.

Again, we thank you and look forward to hearing from you soon concerning an editorial decision.

Yours sincerely,

David Patrick Kateete Ph.D.

Makerere University College of Health Sciences

Kampala, Uganda

POINT-BY-POINT RESPONSE TO THE REVIEWERS’ COMMENTS

SPECIFIC EDITORIAL COMMENTS:

1. The rational in abstract should be strengthened.

Response:

The rationale in abstract has been strengthened, see lines 19 to 28

2. Line 44 and 47, please use Staphylococcus aureus on line 44 and use S. aureus on line 47.

Response:

As advised, we have used Staphylococcus aureus and used S. aureus thereafter, lines 21 and 23.

3. Line 56, please spell out SCCmec since this is the first time it was mentioned.

Response:

As advised, we have spelled out SCCmec at first time of use, see line 75/76.

Journal Requirements:

1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was suitably informed and (2) what type you obtained (for instance, written or verbal). Since your study included minors under age 18, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information.

Response:

We affirm that our manuscript meets PLOS ONE's style requirements stipulated above, including those for file naming. We have also provided additional details regarding participant consent, see lines 96 to 104.

REVIEWERS' COMMENTS

REVIEWER #1:

Comment(s):

In this work, Kateete and colleagues determined the species and the antibiotic susceptibility of Staphylococci isolated from 764 healthy children under 5 years of age residing at the Iganga/Mayuge Health & Demographic Surveillance Site (IMHDSS) in Eastern Uganda. They isolated 513 staphylococci strains belonging to 13 different species. The most prevalent was Staphyloccoccus aureus, followed by S. epidermidis (25,5%), S. heamolyticus (2,3%), S. hominis (0,8%), and S. haemolyticus/lugduniensis (0.58%). The staphylococcal carriage rates from healthy children in rural Eastern Uganda were comparable to those from other countries showing a high level of virulence and AMR genes particularly in S. aureus strains.

Although restricted to a specific area in Africa, the identification of virulent staphylococci may offer some relevant information to the local clinical community, especially with the widespread occurrence of multidrug-resistant organisms and the increasing number of immunocompromised persons in the population. Continuous epidemiological surveillance would also contribute to reducing the Staphylococcal infection burden, hospital stay, and infection morbidity.

The manuscript is a well written, organized, and thorough presentation of the topic. I enjoyed reading the article and found it both relevant in its possible implications for the prevention and the development of targeted treatments and preventive strategies against invasive strains in children. There are some minor typos throughout the text, which can be easily corrected.

Response:

Thank you for the good remarks on our manuscript. We are delighted to hear that you enjoyed reading the manuscript, and that you found it informative regarding the widespread occurrence of antimicrobial resistance in context of immunocompromised individuals.

As advised, we have reworked the manuscript and eliminated the typos. The revised manuscript reads much better now.

REVIEWER #2:

Comment:

This study reports the prevalence of staphylococcal nasopharyngeal colonisation amongst healthy children in Eastern Uganda. 764 children were included, and the authors identified the species of each isolate, their antimicrobial susceptibility and also tested for some virulence and resistance genes.

The sampling appears to have been at a single time point, so “colonisation” should be used rather than “carriage” – which suggests sampling longitudinally. Secondly, only the nasal pharynx was sampled – so “colonisation” should be “nasopharyngeal colonisation” throughout.

Response:

We thank you for this and all your invaluable comments. As advised, we have now used the term “nasopharyngeal colonisation” throughout the text.

Comment:

Acronyms should be defined when they are first used. MDR defined line 170 – but used prior. A definition of MDR needs to be provided in the methods.

Response:

All acronyms are now defined at first use. A definition of multidrug resistance (MDR) has been provided in the methods (lines 130 to 131). We thank you.

Comment:

Proportions have been provided to 1, 2, and 0 decimal places. This needs to be consistent, and I would recommend using whole numbers only. P values should be reported as <0.05 (and or <0.001). There is no section to explain what tests were used to derive P values – nor what hypotheses were actually being tested. In some instances I don’t think the hypothesis testing is useful – e.g. it would be more useful to the reader to list the % of each species resistant to each antibiotic than provide the low P values, which is less informative. The statistical analysis needs to be described in the methods.

Response:

As advised, in the revised manuscript proportions have been provided consistently. Again, as you advised, we have used whole numbers only, and p-values are reported as suggested. Statistical analysis has been provided in the Methods section (lines 158-162), and it includes the specific tests we used to derive the p-values. We have used hypothesis testing only where we thought it would be useful. In Table 4 (pages 15/16) , we listed the percentages (%) in brackets for each species or isolates that was/were resistant or carrying a gene to each antibiotic. We thank you.

Comment:

How did the detection of resistance genes relate to the AST?

The genes listed should be consistent – LukSF or PVL is used (LukSF is more correct).

Response:

The genes investigated are now listed are now consistently used.

As you advised, we have used LukSF instead of PVL.

Regarding detection of resistance genes, apart from the mecA gene that correlated well with phenotypic methicillin/penicillin resistance, the detection of antibiotic resistance genes generally correlated poorly with antibiotic resistance. For example, of the 62 isolates that harbored the genes encoding the aminoglycoside-modifying enzymes (AMEs) (i.e. any of the aac(6')-Ie-aph(2'')-Ia, ant(4')-Ia and aph(3')-IIIa genes), only five (8%) were phenotypically resistant to gentamicin (an aminoglycoside). Additionally, although the vanA gene was detected in 20 isolates, phenotypic vancomycin resistance was not detected. Please note that such findings/observations have been reported before i.e. the detection of resistance genes may not always correlate with phenotypic resistance. We have added these additional aspects to the Results section and discussed the potential their implications in the Discussion section (see lines 241-256; 349-374). We thank you.

Comment:

From line 213 the isolates are compared to another set of isolates. There is no introduction as to where these isolates came from, apart from saying that they were from a hospital. Unless a lot of further information is provided about the provenance of these isolates and why comparison with the study isolates is important or interesting then this section should be removed.

Response:

Many thanks for raising this concern.

The reason why we compared isolates from the IMHDSS (current study) with previously characterized isolates from the hospital was an attempt to identify key differences in the distribution of virulence and antibiotic resistance genes, which could be used to distinguish between invasive/virulent and non-invasive staphylococci. We have clarified this, and we reworked the manuscript to this effect by providing additional information (see lines 82-93; 141-162; and 376-415). The is now an introduction as to where the isolates came from (lines 264-267 and earlier in the methods, page 7). This analysis was insightful as it showed that in Uganda, virulence and resistance genes are more prevent in hospital-associated isolates especially S. aureus at Mulago hospital compared to isolates from the community.

Comment:

In line 126 the genus name does not require italics.

Response:

The genus name has been un-italicized here, and throughout the manuscript. Thank you.

Comment:

Line 143 “MRS staphylococcus” is not correct

Response:

We agree. The phrase “MRS staphylococcus” is not correct and it has been revised, see lines 184-186. Thank you.

Comment:

MRS not defined until line 157.

Response:

This was an oversight. MRS is now defined in the Methods section where it is first used, see line 130.

Comment:

Line 276/277 virulence genes and resistance genes confused

Response:

Many thanks for pointing out this error. We have now separated virulence from resistance genes, please see lines 376 to 387.

Comment:

Line 280 does not with the preceding sentence.

Response:

This section has been clarified and it is now concordant, see lines 377-380. Thank you

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

18 Dec 2019

PONE-D-19-21368R1

Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda

PLOS ONE

Dear Dr. Kateete,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’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.

Both reviewers agreed that the revised manuscript show significant improvement, however, there are still some issues/concerns that need to be addressed.  Please review and address all comments from reviewer #2.

We would appreciate receiving your revised manuscript by Feb 01 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

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)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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: No

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

PLOS ONE 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: 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: The authors have addressed all my suggestions. I found their responses quite satisfactory and the revised version has been much improved. I now recommend the paper for publication with no revisions.

Reviewer #2: The addition of some extra information about the hospital strains was helpful – but it is still inadequate. The methods needs to include information of how these were obtained, who from, what diseases they caused etc. If they’re from adults then careful explanation needs to be made to explain why a study of colonising strains from children would be compared to strains from a hospital obtained from adults. The two strain collections don’t really add up – all staphs vs. S. aureus and S. epi only. (My personal opinion is that this comparison actually doesn’t add a great deal to your manuscript).

Line 57 … S. aureus strains are overwhelmingly coagulase-positive, some are atypical in that they do not produce coagulase.

This requires a reference to be added – ideally several references.

Line 91 … “with the assumption that hospital-associated isolates are more invasive/virulent and/or resistant to antibiotics compared to community-associated isolates.”

Why did you have this assumption? – prior research has shown that most hospital acquired S. aureus infections are endogenous – caused by the carriage strain. This might best be stated as the null hypothesis.

Table 4 still contains P values with a lot of decimal points, as does the text. Use 1 d.p. or use <0.05; <0.001. “S. haemolyticus was significantly more resistant to cefoxitin (p=0.0055), tetracycline (p=0.0041), SXT (p<0.0001), erythromycin (p<0.0001)…”

should become:

“S. haemolyticus was significantly more resistant to cefoxitin (p<0.05), tetracycline (p<0.05), SXT (p<0.001), erythromycin (p<0.001)”

These P values are low – but they don’t help the reader understand the message. It would be better to provide the proportions. These are NOT provided in table 1.

So the text would read:

“Resistance among S. aureus isolates to cefoxitin (x/y, z%), tetracycline (x/y, z%)…..was less common than S. haem isolates (cefoxitin a/b, c% resistant; tetracycline….).

This type of information would be better in a table. Again – I don’t think the chi-sq testing is useful.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: Yes: S R Ritchie

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

12 Jan 2020

January 12, 2020

The Academic Editor, PLOS ONE

Dear Dr. Baochuan Lin, Ph.D.

RE: Submission of revised PONE-D-19-21368R1: Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda

We thank you again for handling our manuscript through the peer-review process, and finding it suitable for publication in PLOS ONE. We carefully considered and effected all the insightful comments from the peer-reviewers, especially Reviewer #2. We hope that you will find our revised manuscript suitable for publication in your esteemed journal. For a detailed point-by-point response to reviewers, please see over.

Looking forward to hearing from you soon concerning an editorial decision.

Yours sincerely,

David Patrick Kateete Ph.D.

Makerere University College of Health Sciences

Kampala, Uganda

POINT-BY-POINT RESPONSE TO THE REVIEWERS’ COMMENTS

SPECIFIC EDITORIAL COMMENT:

Both reviewers agreed that the revised manuscript show significant improvement, however, there are still some issues/concerns that need to be addressed. Please review and address all comments from reviewer #2.

RESPONSE:

We have fully effected all the comments from Reviewer #2.

REVIEW COMMENTS TO THE AUTHOR:

COMMENT:

Reviewer #1: The authors have addressed all my suggestions. I found their responses quite satisfactory and the revised version has been much improved. I now recommend the paper for publication with no revisions.

RESPONSE:

We are delighted to hear so. Thank you for taking time to review the manuscript.

COMMENT:

Reviewer #2: The addition of some extra information about the hospital strains was helpful – but it is still inadequate. The methods needs to include information of how these were obtained, who from, what diseases they caused etc. If they’re from adults then careful explanation needs to be made to explain why a study of colonising strains from children would be compared to strains from a hospital obtained from adults. The two strain collections don’t really add up – all staphs vs. S. aureus and S. epi only. (My personal opinion is that this comparison actually doesn’t add a great deal to your manuscript).

RESPONSE:

In the current revision, we have heeded your advice and removed the comparison between previously characterized hospital isolates and the current study. We thank you for the direction.

COMMENT:

Line 57 … S. aureus strains are overwhelmingly coagulase-positive, some are atypical in that they do not produce coagulase.

This requires a reference to be added – ideally several references.

RESPONSE:

References (See 5-11) have been added to this effect. Thank you.

COMMENT:

Line 91 … “with the assumption that hospital-associated isolates are more invasive/virulent and/or resistant to antibiotics compared to community-associated isolates.” Why did you have this assumption? – prior research has shown that most hospital acquired S. aureus infections are endogenous – caused by the carriage strain. This might best be stated as the null hypothesis.

RESPONSE:

In this revision, we heeded your advice and removed the comparison between previously characterized hospital isolates and the isolates in the current study as it was confusing. Thank you.

COMMENT:

Table 4 still contains P values with a lot of decimal points, as does the text. Use 1 d.p. or use <0.05; <0.001. “S. haemolyticus was significantly more resistant to cefoxitin (p=0.0055), tetracycline (p=0.0041), SXT (p<0.0001), erythromycin (p<0.0001)…” should become: “S. haemolyticus was significantly more resistant to cefoxitin (p<0.05), tetracycline (p<0.05), SXT (p<0.001), erythromycin (p<0.001)”. These P values are low – but they don’t help the reader understand the message. It would be better to provide the proportions. These are NOT provided in table 1. So the text would read: “Resistance among S. aureus isolates to cefoxitin (x/y, z%), tetracycline (x/y, z%)…..was less common than S. haem isolates (cefoxitin a/b, c% resistant; tetracycline….). This type of information would be better in a table. Again – I don’t think the chi-sq testing is useful.

RESPONSE:

We have revised the text as advised. We have provided proportions, and removed the p-values from the results as they are inconsequential. This information is summarized in Table 3, which we have cited in the results section. We hope that the text is now easier to understand. We thank you.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

29 Jan 2020

Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda

PONE-D-19-21368R2

Dear Dr. Kateete,

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

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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 onepress@plos.org.

With kind regards,

Baochuan Lin, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

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 #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

**********

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

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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 #2: Yes

**********

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

PLOS ONE 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 #2: Yes

**********

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 #2: Thank you - I found this revised paper to be more succinct and streamlined. The parts that were confusing have been removed.

**********

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.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: S R Ritchie

5 Feb 2020

PONE-D-19-21368R2

Species and drug susceptibility profiles of staphylococci isolated from healthy children in Eastern Uganda

Dear Dr. Kateete:

I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Baochuan Lin

Academic Editor

PLOS ONE