Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: Emergence of new variant of fimH27 subclone.

Multidrug-resistant Escherichia coli ST131 fimH30 responsible for extra-intestinal pathogenic (ExPEC) infections is globally distributed. However, the occurrence of a subclone fimH27 of ST131 harboring both ExPEC and enteroaggregative E. coli (EAEC) related genes and belonging to commonly reported O25:H4 and other serotypes causing bacteremia in African children remain unknown. We characterized 325 E. coli isolates causing bacteremia in Mozambican children between 2001 and 2014 by conventional multiplex polymerase chain reaction and whole genome sequencing. Incidence rate of EAEC bacteremia was calculated among cases from the demographic surveillance study area. Approximately 17.5% (57/325) of isolates were EAEC, yielding an incidence rate of 45.3 episodes/105 children-years-at-risk among infants; and 44 of isolates were sequenced. 72.7% (32/44) of sequenced strains contained simultaneously genes associated with ExPEC (iutA, fyuA and traT); 88.6% (39/44) harbored the aggregative adherence fimbriae type V variant (AAF/V). Sequence type ST-131 accounted for 84.1% (37/44), predominantly belonging to serotype O25:H4 (59% of the 37); 95.6% (35/44) harbored fimH27. Approximately 15% (6/41) of the children died, and five of the six yielded ST131 strains (83.3%) mostly (60%; 3/5) due to serotypes other than O25:H4. We report the emergence of a new subclone of ST-131 E. coli strains belonging to O25:H4 and other serotypes harboring both ExPEC and EAEC virulence genes, including agg5A, associated with poor outcome in bacteremic Mozambican children, suggesting the need for prompt recognition for appropriate management.

Introduction

Escherichia coli is a common cause of community and hospital-acquired bacterial infection, causing a wide range of clinical diseases and associated with high morbidity and mortality worldwide [1,2]. Two major groups of pathogenic E. coli—diarrheagenic E. coli (DEC) and extra-intestinal pathogenic E. coli (ExPEC)—are recognized, differing in their virulence factors and associated clinical syndromes [2]. ExPEC strains are among the major causes of urinary tract infections (UTI) and/or hospital/community-acquired bacteremia in both industrialized [3,4] and low-income countries [4-6]. These strains differ from DEC or commensal E. coli strains with respect to their virulence factors, with the former requiring specific attributes to cause invasive disease, e.g. the ability to survive in serum, efficient iron uptake mechanisms and internalization by the host [7].

In contrast, six well recognized pathotypes of DEC (enteropathogenic E. coli [EPEC], Shiga toxin (Stx)-producing E. coli [STEC], enterotoxigenic E. coli [ETEC], enteroinvasive E. coli [EIEC], enteroaggregative E. coli [EAEC] and diffusely adherent E. coli [DAEC]) [2], are among the leading etiological agents of childhood [8,9] and travelers’ [10] diarrhea. Among the ExPEC, a wide range of pathogenic lineages of E. coli have been reported, including sequence type (ST) 131, among others [11,12]. ST131 firstly reported in the early 2000s, is a globally disseminated multidrug resistant (MDR) clone with serious clinical impact [13-15], particularly in Africa [12,16]. The most prevalent subclones of ST131 include fimH30 [14], belong to serotypes O25:H4 and O16:H4 [13] is widely reported including clonal diversity [16]. However, to our knowledge, the occurrence of ST131 subclone fimH27, exhibiting serotypes different from O25:H4 or O16:H4 in addition to harboring EAEC genes (e.g. aggR and AAF/V), remains obscure.

EAEC has long been regarded as an intestinal pathogen and therefore unlikely to cause disease in normal patients outside of the intestinal tract [17]. However, recent reports have linked EAEC strains with urinary tract infection [18] and fatal hemolytic uremic syndrome [19] outbreaks. The role of EAEC in extra-intestinal infections and associated outcomes among African children admitted to hospitals, remains unknown. Through our ongoing invasive bacterial disease surveillance, we previously documented E. coli as among the top five pathogens associated with community-acquired bacteremia in Mozambican children, with an associated case fatality ratio of nearly 10% [6]. Thus, assessing the molecular virulence markers of E. coli strains circulating is an important first step in understanding the molecular epidemiology of this entity in Mozambique, with the hope of informing appropriate control or prevention strategies. Herein, we aim to characterize the molecular epidemiology of E. coli causing childhood bacteremia in a rural Hospital in Mozambique between 2001 and 2014.

Materials and methods

Ethics statement

Clinical data were routinely collected from an ongoing morbidity surveillance system in Manhiça district health facilities, established as part of CISM’s HDSS and approved by the Mozambican Ministry of Health. All residents of the district of Manhiça have signed an individual informed consent to become part of the ongoing HDSS established in the area.

Study population

The study was conducted by the “Centro de Investigação em Saúde de Manhiça (CISM)” at the Manhiça District Hospital (MDH), the main referral health facility for the Manhiça district, a rural area located 80 km north of Maputo, southern Mozambique. The district has an estimated population of 183,000 inhabitants, and in this area, CISM has been running a continuous health and demographic surveillance system (HDSS) since 1996, currently covering the entire district’s population. A full description of the geographical and socio-demographic characteristics of the study community has been presented elsewhere [20]. Of importance, HIV sero-prevalence in the area is among the highest in the world (40% of the general adult population) [21]. CISM is adjacent to the MDH, and since 1997, the hospital and CISM have jointly operated a 24-hour surveillance of all pediatric (<15 years of age) visits to the outpatient department and admissions to the wards including surveillance of invasive bacterial disease as described previously [6].

Sample collection and laboratory procedures

As part of routine clinical practice at MDH, a single venous blood specimen for bacterial culture was systematically collected upon hospital admission for all children <2 years of age, and for children aged 2 to <15 years with axillary temperature ≥39.0°C or with signs of severe illness as judged by the admitting clinician for bacterial isolation as detailed described elsewhere [6].

Detection of diarrheagenic E. coli pathotypes, phylogenetic groups and virulence factors

Three hundred and twenty-five frozen E. coli isolates recovered from blood cultures were retrieved, sub-cultured on MacConkey, and screened for the presence of EAEC, ETEC, EPEC, and STEC markers by multiplex polymerase chain reaction (PCR) [22] including phylogenetic group as described elsewhere [23]. Additionally, we tested the isolates for various ExPEC and DEC virulence genes by conventional multiplex PCRs, targeting 44 genes including those commonly prevalent in EAEC (such as aggR, aatA, aap, aaiC and the recently discovered aar gene) [24,25]. Positive samples were confirmed by sequencing five isolates for each gene of interest in the Sequencing Core Facility of the University Of Virginia School Of Medicine, Charlottesville, VA, USA.

Antimicrobial resistance and mechanisms of resistance

The antimicrobial susceptibility phenotype for ampicillin, amoxicillin-clavulanic acid, cefuroxime, ceftriaxone, cefotaxime, aztreonam, ertapenem, imipenem, meropenem, nalidixic acid, ciprofloxacin, chloramphenicol, amikacin, tobramycin, Gentamicin, Tetracycline and trimethoprim-sulfamethoxazole (SxT) was determined by a conventional disk diffusion method on Mueller Hinton agar [26] using commercially available disk (Oxoid, Basingstoke, Hampshire, UK). The interpretative category of resistance was determined according to the Clinical Laboratory Standard Institute (CLSI) guidelines [27]. Multidrug resistance was defined as resistance to three or more unrelated antibiotic families and we considered non-susceptible isolates those with an intermediate or full resistant profile [28]

Serotyping and whole genome sequence (WGS)

Somatic (O) and flagella (H) antigens were phenotypically identified using commercially available antisera as described elsewhere [29,30]. The following designations were included: ‘‘O rough,” the boiled culture auto-agglutinated, suggesting absence of O antigen; ‘‘O?” when it could not be determined whether the strain produces an O antigen (precipitation with Cetavlon indicates an acidic polysaccharide that could represent capsular K antigen); and ‘‘ONT,” when the O antigen was found to be present but could not be typed. Serotyping was performed on all bloodstream isolates positive for EAEC markers and a subset of other E. coli bacteremic strains at the International Escherichia and Klebsiella Centre, Department of Bacteria, Parasites and Fungi, Statens Serum Institut (SSI), Copenhagen, Denmark. Additionally, serotypes were also assessed by WGS and compared to conventional serotyping.

Forty-four EAEC isolates (the total of positive by PCR) and 22 non-EAEC isolates for control purpose (randomly selected) were sequenced by using Illumina Miseq (Illumina, San Diego, CA, USA). Briefly, Genomic DNA from isolates was purified using Qiagen DNeasy Blood and Tissue kit (Qiagen, Valencia, USA) according to the kit protocol. Initial DNA concentrations were measured and quantified using Qubit Flourometer and dsDNA BR/HR Assay Kit (Thermo Fisher Scientific). Sample and library preparation were performed using the Nextera XT v2 DNA library Preparation Kit (Illumina, Sand Diego, USA). Libraries were finally purified by Agencourt AMPpure XP System (Beckman Coulter, Indianapolis, USA). WGS data were pre-processed employing a QC-pipeline (available at https://github.com/ssi-dk/SerumQC), where isolate sequences were removed in case of contamination with more than 5% of another genera, as well as sequences representing EAEC isolates with genome sizes outside the range of 4.64 Mbp-5.56 Mbp. Isolate sequences were removed from the dataset if assemblies comprised of more than 350 contigs. De novo assemblies were carried out using CLC Genomics Workbench 10 with a minimum contig length of 200 bp. The genome size, N50, and contigs are presented in S1 Table.

Sequence type, in silico serotype and virulence genes were determined from the de novo assembled genomes using the webtools available at https://cge.cbs.dtu.dk/services/ [31]. The least ambiguous phenotypical or in silico serotype was used in the final analysis i.e. non-motile strains (H-) were given the in silico determined fliC H type, the in silico O type was used on O rough and O? and the phenotypic O group was used if the in silico O type was ambiguous or non-typeable.

Definitions

Bacteremia was defined as the isolation of at least one non-contaminant bacteria from the blood culture collected on admission. Bacteremic EAEC strains were defined as isolates from blood culture testing positive for one of the following genes: aggR, aaiC or aatA genes by multiplex PCR. Other bacteremic E. coli were defined as E. coli strains from blood culture excluding EAEC, EPEC, ETEC and STEC.

Case fatality ratios (CFR), represent in-hospital mortality due to bacteremia calculated for admitted children with known outcome (i.e. discharged alive, or dead), excluding patients that left the hospital without medical permission or were transferred to Maputo Central Hospital as previously described [6].

Statistical analysis

Statistical analyses were performed using STATA software, version 13.0 (Stata Corp., College Station, TX, USA). The proportion of virulence factors found among children infected with EAEC and other bacteremic E. coli was compared using Chi-squared or Fisher’s exact tests as appropriate. Minimum community-based incidence rates (MCBIRs) for E. coli bacteremia (EAEC and other bacteremic E. coli excluding ETEC and EPEC) were calculated referring cases to population denominators establishing time at risk (child years at risk [CYAR]) inferred from the HDSS census information. Children did not contribute to the numerator or denominator for a period of 15 days after each episode or when they were outside the study area.

CART analysis was also performed, as previously described [24] (CART Pro Version 7.0; Salford Systems). We included the collective number of virulence genes present (virulence factor score, VFS) in putting 48 factors of interest as binary (present/absent) independent predictive variables along with a continuous ‘‘factor total” that was a sum of all factors including the presence of malaria. Alive/death was the binary dependent outcome variable for isolates causing bacteremia. Furthermore, the CART analyses for bacteremic EAEC were also assayed for 66 genes assessed by WGS.

Results

Screening for EAEC markers, serotypes and phylogenetic groups distribution

From January 2001 to December 2014, 37,536 blood cultures were collected from children younger than 3 years of age admitted to the MDH; and 325 (0.8%) were positive for E. coli. Of these, 57 (17.5%) met the definition of EAEC, 6 (1.8%) ETEC and 2 (0.6%) EPEC; while the remaining 260 (80.0%) were classified as other bacteremic E. coli. Children with EAEC bacteremia appeared to be younger than those with bacteremia secondary to other E. coli [mean: 10.1 months (SD = 7.2) vs. 12.4 (SD = 8.3), p = 0.057], albeit not statistically significance.

Sixty-six (20.3%) [44 EAEC and 22 other bacteremic E. coli] of the 325 E. coli isolates were serotyped, with serotype O25:H4 being the most frequent, accounting for 22 isolates (33.3%), followed by O127:H4 with six (9.1%) isolates, and O51:H4 and O86:H4 with four (6.1%) isolates each. All O25:H4, five of six O127:H4, and all O51:H4 and all O86:H4 were EAEC bacteremic E. coli isolates, followed by nine isolates (13.6%) of seven different serotypes (Table 1).

Table 1

Genomic characteristics of the 44 enteroaggregative E. coli strains isolated from children admitted at the MDH with bacteremia, analyzed by WGS.

C-number	Phenotypical serotype	Molecular serotype	ST	Fimtype	Virulence genes	AAF	Phylogeny	Age*	Outcome	Malaria	Diarrhea	Sex
C169-15	O125ab/O176:H-	O176:H33•	10	H54	aaiC,astA,capU,gad,mchB,mchC,mchF,mcmA,ORF3,ORF4,pic,sat, fyuA, hra, iutA, agn43	Neg.	A	10	Alive	UNK	Yes	F
C194-15	O125ab/O176:H-	O176:H33•	10	H54	aaiC,astA,capU,gad,mchB,mchC,mchF,mcmA,ORF3,ORF4,pic,sat,fyuA, hlyA, hra, iutA, agn43	Neg.	A	12	Alive	Yes	No	F
C190-15	O11:H-	O11:H18•	31	Neg.	aap,aar,aatA,aggA,aggB,aggC,aggD,aggR,air,astA,eilA,gad,iha,iss,lpfA,ORF3,ORF4,sat, fyuA, traT, papC, papA, hlyA, hra, papGII, iutA, agn43	I	B2	9	Alive	Yes	No	F
C188-15	O153:H30	H30•	38	H5	aap,aar,aggA,aggB,aggC,aggD,aggR,capU,eilA,gad,iss,nfaE,ORF3,ORF4, fyuA, traT, hra, agn43	I	D	3	Alive	Yes	Yes	F
C175-15	O44:H34	O17/O77:H34•	130	H47	aap,aatA,agg4A,agg4C,agg4D,aggR,air,eilA,gad,lpfA,ORF3,ORF4,sepA, agn43	IV	D	9	Alive	Yes	No	M
C159-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, papA, hlyA, hlyC, papGII, iutA, agn43	V	B2	16	Alive	No	No	M
C164-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	13	Alive	No	No	M
C166-15	Orough:H4	O127:H4•	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	30	Alive	Yes	No	M
C167-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	28	Alive	No	No	M
C168-15	O?:H4	O51:H4•	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, papGII, iutA, agn43	V	B2	9	Alive	No	No	F
C170-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyC, papGII, iutA, agn43	V	B2	4	Dead	No	Yes	M
C171-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iha,iss,nfaE,ORF4,sat, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	9	Alive	Yes	Yes	M
C174-15	O86:H4	O86:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA	V	B2	3	Alive	Yes	No	M
C177-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	8	Alive	No	No	M
C178-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyC, aer, papGII, iutA, agn43	V	B2	9	Alive	No	Yes	M
C179-15	O127:H4	O127:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,iss,ORF3,ORF4, fyuA, papA, hlyA, hlyC, aer, papGII, iutA	V	B2	7	Dead	No	No	M
C182-15	O15:H4	O15:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, aer, papGII, iutA, agn43	V	B2	19	Alive	No	No	M
C184-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, papGII, iutA, agn43	V	B2	7	Alive	No	No	F
C185-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF4,pic, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	8	Transf.	Yes	No	M
C189-15	O86:H4	O86:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	32	Dead	No	No	M
C191-15	O127:H4	O127:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, papGII, iutA, agn43	V	B2	13	Left	UNK	Yes	M
C193-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iha,iss,ORF3,ORF4,sat, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	6	Alive	Yes	Yes	M
C197-15	O25:H-	O25:H4•	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	16	Alive	No	Yes	M
C198-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	5	Alive	No	Yes	M
C199-15	O25:H4	O25:H4	131	H27	aap,aar,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	3	Alive	No	Yes	M
C200-15	O51:H4	O51:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, papGII, iutA, agn43	V	B2	10	Alive	No	No	M
C204-15	O86:H4	O86:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA	V	B2	7	Dead	No	Yes	M
C206-15	Orough:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	7	Alive	Yes	Yes	M
C207-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iha,iss,mchB,mchC,mchF,ORF3,ORF4,sat	V	B2	1	Alive	No	Yes	M
C209-15	O86:H4	O86:H4	131	H27	aap,aar,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA	V	B2	21	Alive	Yes	No	M
C210-15	O?:H4	O51:H4•	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	15	Alive	UNK	No	M
C211-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	4	Dead	Yes	Yes	M
C212-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyC, aer, papGII, iutA, agn43	V	B2	10	Alive	Yes	Yes	M
C213-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	8	Alive	Yes	No	M
C215-15	Orough:H4	O127:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, aer, papGII, iutA, agn43	V	B2	22	Alive	No	No	F
C216-15	O127:H4	O127:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA	V	B2	10	Alive	Yes	No	M
C217-15	O18ac:H4	O18ac:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, papGII, iutA, agn43	V	B2	1	Transf.	No	No	M
C218-15	Orough:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, papGII, iutA, agn43	V	B2	7	Alive	No	Yes	M
C222-15	O25:H4	O25:H4	131	H27	aap,aar,aatA,agg3B,agg3D,agg5A,aggR,gad,iha,iss,ORF4,sat, fyuA, traT, papC, papA, hlyA, hlyC, aer, iutA, agn43	V	B2	0	Alive	No	Yes	M
C223-15	O51:H4	H4•	131	H27	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	14	Alive	No	No	M
C165-15	O25:H4	O25:H4	131	Neg.	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	B2	13	Alive	Yes	Yes	M
C202-15	O25:H4	O25:H4	131	Neg.	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,cnf1,gad,iss,ORF3,ORF4, fyuA, traT, papC, papA, hlyA, aer, papGII, iutA, agn43	V	B2	7	Alive	UNK	Yes	M
C205-15	O166:H15	O166:H15	349	H93	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,air,capU,eilA,gad,iha,iss,ORF3,ORF4,sat, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	D	5	Alive	Yes	No	M
C219-15	O166:H15	O166:H15	349	H93	aap,aar,aatA,agg3B,agg3C,agg3D,agg5A,aggR,air,capU,eilA,gad,iha,iss,ORF3,ORF4,sat, traT, papC, papA, hlyA, hlyC, aer, papGII, iutA, agn43	V	D	8	Dead	Yes	No	F

*Age in months; UNK–unknown; and REF- referred to Maputo central hospital; Transf.—Transferred

Definitions: papA, P fimbriae structural subunit; papC, P fimbria assembly; papGII, P fimbria adhesin; fimH, type 1 fimbriae; hra, heat-resistant agglutinin; hlyA, α-hemolysin; hlyC hemolysin, cnf1, cytotoxic necrotizing factor; sat, secreted autotransporter toxin; pic, autotransporter protease; fyuA, yersiniabactin system; aer, aerobactin; iutA, aerobactin receptor; traT, serum resistance associated; ibeA, invasion of brain endothelium; astA, EAEC heat-stable toxin; pet, Plasmid-encoded toxin; sigA, IgA protease-like homolog; pic Serine protease precursor; sepA Shigella extracellular protease; ORF3 ORF4—Cryptic protein; aap, Dispersin, antiaggregation protein; aaiC, AaiC secreted protein; aggR, EAEC transcriptional activator; aatA, Dispersin transporter protein; aggA, AAF/I major fimbrial subunit; aggB, minor fimbrial subunit; aggC, usher; aggD, chaperone; agg4A, AAF/IV major fimbrial subunit; agg4B, minor fimbrial subunit; agg4C, usher; agg4D, chaperone; agg5A, AAF/V fimbrial subunit; agg3B, minor fimbrial subunit; agg3C, usher; agg3D, chaperone; aar, AggR-activated regulator; eilA, Salmonella HilA homolog; capU, Hexosyltransferase homolog; air, Enteroaggregative immunoglobulin; iss, increased serum survival gene, agn43; outer membrane protein; iha, adherence-conferring protein; gad, Glutamate decarboxylase; mchB, mchC, mcmA, genes required for the production of the antimicrobial peptide microcin H47; mchF; microcin transporter protein; nfaE, non-fimbrial adhesion the molecular serotype confirmed the conventional serotype except in seven strains (marked with •)

Virulence factors detected by conventional multiplex PCR

Of the 57 EAEC, detailed virulence factors were assessed in the 44 isolates serotyped and sequenced. Of the 44 EAEC strains serotyped from blood, 41 (93.2%) met the definition of typical EAEC (presence of the master regulator gene, aggR) and were positive for aar (AggR- activated regulator). Only one typical EAEC (aggR+) strain lacked aatA while 37 showed the combined presence of virulence genes aggR, aap, aatA and ORF3; and five and two isolates lacked the aap or ORF3 genes, respectively. Table 2 shows the prevalence of virulence factors comparing EAEC and other bacteremic E. coli isolates analyzed by conventional PCR. Notably, 35 out of 44 (79.5%) of EAEC isolates contained the three genes—iutA, fyuA and traT (associated with ExPEC) similar to 65% (100 isolates) of other bacteremic E. coli. The toxins commonly found in septicemic E. coli (e.g. hlyA, hylC and cnf1) were more prevalent among EAEC isolates compared to other bacteremic E. coli (Tables 2 and 3).

Table 2

Comparison of virulence factors of EAEC causing bacteraemia versus other bacteremic E. coli strains isolated in Mozambican children detected by conventional multiplex PCR–EAEC associated virulence genes.

Virulence Gene	Bacteraemic EAEC (N = 44)n (%)	Other bacteraemic E. coli (N = 164)n (%)	p-value
EAEC
aatA	39 (88.6)	0 (0.0)	<0.001
aaiC	2 (4.6)	0 (0.0)	0.044
aggR	41 (93.2)	0 (0.0)	<0.001
aap	41 (93.2)	12 (7.3)	<0.001
ORF3	39 (88.6)	10 (6.1)	<0.001
ORF4	41 (93.2)	6 (3.7)	<0.001
aar	41 (93.2)	44 (27.3)	<0.001
astA	5 (11.4)	72 (43.9)	<0.001
EAEC adhesins
aafC	0 (0.0)	22 (13.4)	0.005
agg3/4/5C	38 (88.4)	7 (4.2)	<0.001
aggA	2 (4.5)	2 (1.2)	NS
agg3A	0 (0.0)	0 (0.0)	NS
aafA	0 (0.0)	3 (1.8)	NS
agg4A	1 (2.3)	21 (12.8)	0.052
Agg5A	39 (88.6)	7 (4.3)	<0.001

Table 3

Comparison of virulence factors of EAEC causing bacteraemia versus other bacteremic E. coli isolated in Mozambican children detected by conventional multiplex PCR–ExPEC and miscellaneous associated virulence genes.

Virulence Gene	Bacteremic EAEC (N = 44)n (%)	Other bacteremic E. coli (N = 164)n (%)	p-value
ExPEC adhesins
sfaS	0 (0.0)	31 (18.9)	0.002
fimH	41 (93.2)	153 (93.3)	NS
papA	43 (97.7)	96 (58.5)	<0.001
papC	37 (84.1)	132 (80.5)	NS
papGII	38 (86.4)	125 (76.2)	0.033
papGIII	1 (2.3)	3 (1.8)	NS
afa_dra	3 (6.8)	63 (38.4)	<0.001
Class I SPATEs
sat	1 (22.7)	31 (18.9)	NS
pet	0 (0.0)	32 (19.5)	0.001
sigA	0 (0.0)	1 (0.6)	NS
Class II SPATEs
sepA	1 (2.3)	19 (11.6)	NS
pic	4 (9.1)	15 (9.2)	NS
vat	3 (6.8)	59 (36.0)	<0.001
epeA	0 (0.0)	0 (0.0)	NS
eatA	0 (0.0)	3 (1.8)	NS
Siderophores
iutA	41 (93.2)	144 (87.8)	NS
iroN	0 (0.0)	67 (41.1)	<0.001
fyuA	40 (90.1)	137 (83.5)	0.043
aer	30 (68.2)	127 (77.4)	NS
Toxins
hlyA	36 (81.8)	50 (30.4)	<0.001
hlyC	31 (70.5)	70 (42.7)	<0.001
cnf1	27 (61.4)	58 (35.4)	0.002
hra	4 (9.1)	47 (28.6)	0.007
cdtb	0 (0.0)	0 (0.0)	NS
agn43	38 (86.4)	26 (15.9)	0.017
Miscellaneous
air	4 (9.1)	33 (20.1)	NS
eilA	5 (11.4)	85 (52.4)	<0.0001
capU	5 (11.4)	35 (21.6)	NS
ibeA	0 (0.0)	11 (6.7)	NS
traT	38 (86.4)	131 (79.9)	NS

Whole genome sequencing (WGS) of EAEC isolates

WGS data found that the 44 EAEC strains belonged to six different ST types with ST-131 accounting for 84.1% (37/44) of the strains, of which 35 (95.6%) harbor fimH27, while the other two were negative for fimH typing. Furthermore, 59% (22/37) ST131 strains belonged to the serotype O25:H4, while the remaining 41% (15/37) fall into five serotypes [O127:H4 (5 strains), O86:H4 (4), O51:H4 (4), O18ac:H4 (1) and O15:H4 (1)] (Table 1). Additionally, 93.2% (41/44) harbored the EAEC virulence plasmid pAA encoding the master regulator aggR and several aggR- regulated genes, such as aap (dispersin), aatA (dispersin translocator), the newly discovered aggR repressor aar [32], and the aggregative adherence fimbriae (AAF) gene cluster. The AAF/V variant was found in 88.6% (39/44) of the EAEC strains followed by AAF/I (2/44) and AAF/IV (1/44). The sequence analysis confirmed the PCR analysis and the presences of several ExPEC associated virulence genes (iutA, fyuA, traT, hlyA, hylC, cnf1, and papGII). Furthermore, we found the increased serum survival gene—iss in 95.5% (42/44) of the EAEC strains positive for aggR. Lastly, the molecular serotype confirmed the conventional serotype except in seven strains (marked with • in Table 1). Additionally, virotype classification did not provide a clear discrimination of our strains according to Dahbi et. al. [33], suggesting possible occurrence of virotype E sub-type requiring further characterization of isolates.

Antimicrobial susceptibility and associated mechanisms

We also assessed the antimicrobial resistance of the 44 EAEC strains, documenting high prevalence of resistance to the most commonly available and used antibiotic for empirical treatment (ampicillin, gentamicin and chloramphenicol) in our community including multidrug resistance (MDR) 97% as demonstrated in Table 4. WGS also identified genes conferring resistance towards three or more groups of antibiotics; Aminoglycosides, Macrolides, Phenicols, Quinolones, Sulphonamides, Tetracyclines, Trimethoprim, and/or β-Lactams (Table 5).

Table 4

Antimicrobial susceptibility profile of EAEC causing bacteremia in young children in Manhiça District Hospital.

Antibiotic name	No. tested	%R	%I	%S
Ampicillin	33	97	0	3
Amoxicillin/Clavulanic acid	33	21,2	33,3	45,5
Piperacillin/Tazobactam	31	0	0	100
Cefuroxime	33	0	36,4	63,6
Ceftazidime	33	0	0	100
Ceftriaxone	33	0	0	100
Cefoxitin	33	0	3	97
Aztreonam	33	3	0	97
Ertapenem	33	0	3	97
Imipenem	33	3	0	97
Meropenem	33	0	0	100
Amikacin	33	6,1	0	93,9
Gentamicin	33	48,5	0	51,5
Tobramycin	33	24,2	6,1	69,7
Nalidixic acid	33	3	3	93,9
Ciprofloxacin	33	18,2	0	81,8
Trimethoprim/Sulfamethoxazole	33	81,8	0	18,2
Chloramphenicol	33	72,7	0	27,3
Tetracycline	33	39,4	0	60,6

Table 5

Detection by WGS of genes conferring resistance of EAEC causing bacteremia in young children in Manhiça District Hospital.

C-number	ST	Molecular serotype	fimtype	Phylogeny	Resistance
C159-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C164-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C165-15	131	O25:H4	Negative	A	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C166-15	131	O127:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C167-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C168-15	131	O51:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C169-15	10	O176:H33	fimH54	A	aadA1-like,blaOXA-1-like,catA1-like,strA,strB,sul2,tet(B)
C170-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C171-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,aadA2,blaTEM-1B,catA1-like,dfrA12,mph(A),strA,strB-like,sul1,sul2
C174-15	131	O86:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C175-15	130	O17/O77:H34	fimH47	D	Not found
C177-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C178-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C179-15	131	O127:H4	fimH27	A	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C182-15	131	O15:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C184-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C185-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C188-15	38	H30	fimH5	D	aadA1,blaTEM-1B,catA1-like,dfrA1,strA,strB,sul2,tet(D)
C189-15	131	O86:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C190-15	31	O11:H18	Negative	A	blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C191-15	131	O127:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C193-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,aadA2,blaTEM-1B,catA1-like,dfrA12,dfrA14-like,mph(A),strA-like,strB,sul1,sul2
C194-15	10	O176:H33	fimH54	A	aadA1-like,blaOXA-1-like,catA1-like,strA,strB,sul2,tet(B)
C197-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C198-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C199-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C200-15	131	O51:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C202-15	131	O25:H4	Negative	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C204-15	131	O86:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C205-15	349	O166:H15	fimH93	B1	blaTEM-1B,strA,strB-like,sul2
C206-15	131	O25:H4	fimH27	D	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C207-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,aadA2,blaTEM-1B,catA1-like,dfrA12,mph(A),sul1
C209-15	131	O86:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B
C210-15	131	O51:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)
C211-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C212-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C213-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C215-15	131	O127:H4	fimH27	D	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C216-15	131	O127:H4	fimH27	A	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C217-15	131	O18ac:H4	fimH27	B1	aac(3)-IIa,aac(3)-IId-like,blaTEM-131-like,blaTEM-1B,catA1-like,dfrA7,floR-like,strA,strB,sul1-like,sul2,tet(A)-like
C218-15	131	O25:H4	fimH27	B1	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2
C219-15	349	O166:H15	fimH93	A	blaTEM-1B,dfrA8,strA,strB-like,sul2,tet(A)
C222-15	131	O25:H4	fimH27	B2	aac(3)-IId-like,aadA2,blaTEM-1B,catA1-like,dfrA12,sul1
C223-15	131	:H4	fimH27	B2	aac(3)-IId-like,blaTEM-1B,catA1-like,dfrA7,strA,strB,sul1-like,sul2,tet(A)

Burden and clinical impact of EAEC bacteremia

Of the total of 325 E. coli bacteremia episodes, only 127 (39.1%) occurred in children living within the DSS area, yielding an overall incidence rate of E. coli of 110.1 cases/100,000 child-years (95%CI: 92.5–131.0) with the highest incidence occurring among infants (181.1 cases/100,000 child-years; 95%CI: 143.7–228.1) (Table 6). EAEC incidence among infants was 45.3 cases/100,000 child-years (95%CI: 28.5–71.8), peaking in 2002 and 2003 with 71.5 and 78.9 cases/100,000 children-years, respectively (Fig 1).

Table 6

Minimum community-based incidence rates of Escherichia coli bacteemia among children aged less than 3 years living in Manhiça DSS study area, 2001–2014.

Category	Time-at-risk	No. episodes	Incidence rates a (95% CI)
Bacteremic EAEC
0–11 months	39767.38	18	45.3 (28.5–71.8)
12–23 months	38283.89	7	18.3 (8.7–38.4)
24–35 months	37309.93	3	8.04 (2.6–24.9)
All ages	115361.20	28	24.3 (16.7–35.2)
Other E. coli bacteremia
0–11 months	39766.1	54	135.8 (104.0–177.3)
12–23 months	38282.7	37	96.7 (70.0–133.4)
24–35 months	37309.7	8	21.4 (10.7–42.9)
All ages	115358.8	99	85.8 (70.5–104.5)
All bacteremic E. coli
0–11 months	39765.5	72	181.1 (143.7–228.1)
12–23 months	38282.5	44	114.94 (85.5–154.5)
24–35 months	37309.6	11	29.5 (16.3–53.2)
All ages	115357.5	127	110.1 (92.5–131.0)

a Incidence rates were calculated referring cases to population denominators establishing time at risk (child years at risk [CYAR]) inferred from the HDSS census information. Children did not contribute to the numerator or denominator for a period of 15 days after each episode or when they were outside the study area.

Fig 1

Trends of minimum estimates of community incidence rates of E. coli bacteremia during the study period, 2001–2014.

Nutritional status was recorded for 51 of the 57 EAEC patients, of whom 8 (15.7%) were severely malnourished, compared with 70/228 (30.7%) from other E. coli bacteremia group, p = 0.031; and the CFR was similar in the two groups (15.8%, 9/57 vs. 14.7%, 38/258; p = 0.8, for EAEC and other E. coli bacteremia, respectively). Fever (92.9% vs. 89.5%), diarrhea (42.1% vs. 32.5%), vomiting (36.8% vs. 29.1%), and cough (71.9% vs. 73.3%) were found in similar proportion between children infected with EAEC (n = 57) and other E. coli bacteremia (n = 258), respectively.

The Classification and Regression Tree (CART) analysis suggests the presence of 2 clusters associated with poor outcome in the absence of malaria. Cluster 1 comprising strains testing positive for papGII and hra in the absence of sfaS (Node 1) and cluster 2 comprising strains harboring cnf1 in the absence of hra and afa_dr (Node 2) (Fig 2). In addition, we demonstrated the presence of fatal strains harbored hlyC and orf3 genes in the absence of agn43 (Node 1) or belonging to ST131 clone harboring hlyA and aer lacking astA toxin (Node 2) among children infected by EAEC (Fig 3). Case fatality ratio among children infected sequenced EAEC strains was 14.6% (6/41), mostly related to ST131 strains (83.3%; 5/6); and 60% (3/5) children with poor outcome were infected by serotypes other than O25:H4, namely O86:H4 (n = 2) and O127:H4 (n = 1).

Fig 2

Diagram of CART analysis against hospital outcome.

We included the collective number of virulence genes present (virulence factor score, VFS) in putting 48 factors of interest as binary (present/absent) independent predictive variables along with a continuous ‘‘factor total” that was a sum of all factors including the presence of malaria. We identified 2 clusters associated with poor outcome in the absence of malaria: i) comprising strains testing positive for papGII and hra_in the absence of sfaS (node 1); and ii) comprising strains harboring cnf1 in the absence of hra and afa_dr (node 2).

Fig 3

CART analyses for bacteremic EAEC assessing 66 genes by WGS.

Regardless of age or serotype we demonstrated the presence of fatal strains harboring hlyC and orf61 genes in the absence of agn43 (node 1) or belonging to ST131 clone harboring, hlyA and aer lacking astA toxin (node 2).

Classification and regression tree (CART) classification tree topology reveals combinations of factors most strongly associated death in the absence of malaria (Fig 2) or for EAEC strains (Fig 3). We considered all genotypic and phenotypic assays performed: aatA, aggR, aaiC, aap, ORF3, sat, sepA, pic, sigA, pet, astA, aafC, agg3/4C, aafA, agg3A, aggA, agg4A, air, capU, eilA, ORF61. Each branch of the CART tree ends in a terminal ''node'' (blue boxes), and each terminal node is uniquely defined by the presence or absence of a predictive factor such as a gene. The tree is hierarchical in nature.

Discussion

This is the first study conducted in Mozambique characterizing E. coli strains causing childhood bacteremia; documenting the novel subclone of ST131 harboring EAEC genes causing bacteremia in children, with its highest incidence peaking during infancy. During the last years, evidence of involvement of non-fimH30 ST131 isolates and fimH30 subclone isolates fulfilling molecular criteria for EAEC in extra-intestinal infections have been reported [18,34]. However, to our knowledge, this is the first report analyzing overtime trend incidences of EAEC-associated bacteremia in African children, showing a magnitude similar to that caused by Staphylococcus aureus or Hib (pre-vaccine introduction) in our population [6], suggesting that EAEC is currently playing an important role as a cause of childhood E. coli bacteremia in this setting.

The high incidence of EAEC reported here could either be related to pathogen or to host factors, including malnutrition or HIV, both highly prevalent in our study area and also known to enhance translocation of commensal bacteria to the bloodstream [2]. Despite the limitation on HIV data in our study population, we believe that the EAEC incidence reported here is possibly due to properties of the pathogen. If it was favored by HIV or malnutrition co-infection, we would expect to also find a high prevalence of the other pathotypes (e.g. EPEC or ETEC) also prevalent among healthy children [35].

As a common enteric isolate, we hypothesize that extra-intestinal EAEC may arise via the transfer the pAA plasmid more classical invasive pathogens, thus transferring additional virulence traits. This is supported by the high prevalence of classical ExPEC virulence genes within our EAEC, such as hlyA which is known to induce oxidative stress in blood [25], and which is also associated with polymorphonuclear lysis/necrosis and lung injury in vivo in a rat model of E. coli pneumonia [36]. The low prevalence of the chromosomal aaiC gene in our strains compared to aggR and aatA on the pAA plasmid may be additional support for transfer of the pAA plasmid. It also suggests that aaiC is not a good marker for EAEC bacteremic strains in our community.

The high prevalence of adhesion AAF/V in our strains is noteworthy, suggesting a high degree of phylogenetic relatedness of our strains. This is underscored by the presence of papC or type 1 fimbriae (fimH) in more than 90% of isolates, suggesting that these strains may derive from urinary tract infections (UTI) strains, despite the lack of clinical information with regard to diagnosis of UTI. The change in fimH alleles might improve colonization abilities of the different clades (global dissemination of a multidrug resistant E. coli clone) [33], however, to our knowledge this is second report of fimH27 subclade, which has recently been reported in ST405 accounting for 13% of ExPEC strains isolated from clinical isolates in Nigeria [12]. In contrast to the fimH30 subclade that is characterized to be resistant to extended spectrum of β-lactamase or fluoroquinolones [15,37,38], our strains were susceptible to third generation cephalosporines and fluoroquinolones. However, the resistance profile of our fimH27 strains was similar to those reported by Roer et al from bloodstream infections in Denmark [39], despite the high serotype- and ST diversity in the latter. Interestingly, the fact that the isolates circulating in our community do not fit in the classical classification of virotypes [33] supports the hypothesis of the presence of a new entity that require further characterization. Plasmid analysis of our strains compared to globally disseminated fimH30 and fimH27 of ST405 is underway and will be published elsewhere.

Also notable is the fact that WGS identified the presence of the iss gene, recognized for its role in ExPEC virulence and considered a distinguishing trait of avian ExPEC but not of human ExPEC [40], suggesting that some strains may fit in the classification of avian pathogenic E. coli (APEC).

In addition, both serotyping and WGS data support the presence of serotype O25:H4 clone ST131, a clone significantly associated with urinary tract infections and bacteremia [37]. More interesting is the finding that 15 out of 37 (41.7%) of ST131 strains were from distinct serotypes from the traditional O25:H4 and to our knowledge never previously reported: O127:H4 (5 strains), O51:H4 (4), O86:H4 (4), O18ac:H4 and O15:H4 [41,42]. EPEC has been shown to cluster in related groups sharing the H antigen (H2 and H6) that differ only on the O antigen, which might suggest that the LPS operon may be located in a phage region and can be transferred by transduction among EPEC [43]. Here, we find that these strains share H4 but differ in their O antigen, yet belong to the same MLST type ST131, a finding that certainly warrants further investigation. Importantly, E. coli phylogenetic analyses have generally attached greater significance to the H antigen as a marker of shared genetic ancestry, suggesting that the high preponderance of H4 strains in Mozambique may indeed signal the existence of a highly virulent and longstanding pathogen. Further epidemiologic studies should address the importance of H4 flagellar clones.

The WGS analysis illustrated that the recently discovered Aar, that has been hypothesized to act directly or indirectly as a virulence suppressor, modulating virulence because of selection towards clinical attenuation, was present in almost all isolates. These data may also strongly support an important role for the aar gene in E. coli epidemiology; similar to what was found in previous studies in Mali and Brazil where aar-negatives AAF/IV variant showed increased pathogenicity [24,44].

Interestingly CART analysis data showed that deaths are likely to occur in the absence of malaria; nearly 20% of such children died, of which 12 when infected with strains harboring papGII and eight in those testing positive for hra (Fig 2, node 1). This finding reinforces the need of routine screening of bacterial pathogens among children admitted in developing countries where most of deaths are attributable to malaria due to the limited microbiology infrastructure. Indeed, fatal EAEC strains are also related to the presence or absence of specific virulence factors found in ST131 strains (Fig 3) with attributable case fatality greater than that caused by invasive non-typhoidal Salmonella [45] or S. aureus. In addition, despite the small number of isolates, the poor outcome of ST131 non-O25:H4 serotypes may suggest that those are more virulent that the classical serotype O25:H4, and require further in vitro or in vivo testing to establish its potential virulence [13]. Unfortunately due to the lack of adequacy or incomplete data on appropriate empirical treatment in terms of number of doses and days, the variables of antimicrobial resistance, HIV treatment were not included in the CART analysis which may help to elucidate the relationship of strains virulence profile and poor outcome

Our study sheds light on the etiology of the bacteremia events, suggesting that not only O25:H4 EAEC, but also other previously undescribed EAEC serotypes of ST131 clone strains can cause clinically severe invasive bacteremia in neonates and young children resulting in hospitalization and death in Southern Mozambique, requiring prompt recognition for appropriate management.

Distribution of distribution of contig size lengths, genome size and N50s of the EAEC sequenced strains.

(XLSX)

Click here for additional data file.

11 Feb 2020

Dear Dr. Mandomando,

Thank you very much for submitting your manuscript "Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: emergence of new variant of fimH27subclone" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

His manuscript is interesting and novel, but before being accepted it needs to be improved taking into account the most important comments made by the three reviewers.

In addition, you should compare your results with those found by Roer et al in Denmark.

WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

Roer L, Hansen F, Thomsen MCF, Knudsen JD, Hansen DS, Wang M, Samulioniené J, Justesen US, Røder BL, Schumacher H, Østergaard C, Andersen LP, Dzajic E, Søndergaard TS, Stegger M, Hammerum AM, Hasman H.

J Antimicrob Chemother. 2017 Jul 1;72(7):1922-1929. doi: 10.1093/jac/dkx092.

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

His manuscript is interesting and novel, but before being accepted it needs to be improved taking into account the most important comments made by the three reviewers.

In addition, you should compare your results with those found by Roer et al in Denmark.

WGS-based surveillance of third-generation cephalosporin-resistant Escherichia coli from bloodstream infections in Denmark.

Roer L, Hansen F, Thomsen MCF, Knudsen JD, Hansen DS, Wang M, Samulioniené J, Justesen US, Røder BL, Schumacher H, Østergaard C, Andersen LP, Dzajic E, Søndergaard TS, Stegger M, Hammerum AM, Hasman H.

J Antimicrob Chemother. 2017 Jul 1;72(7):1922-1929. doi: 10.1093/jac/dkx092.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: Objectives and methods are appropiated.

Reviewer #3: Study design partially appropriate

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: Results are described in a logical and straightforward manner.

Reviewer #3: results section needs clarification

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: The discussionand conclusions provides a balanced assessment of the author’s findings.

Reviewer #3: only partially

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Editorial and Data Presentation Modifications?

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Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: (No Response)

--------------------

Summary and General Comments

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Reviewer #1: This study examines E. coli ST131 causing bacteremia in Mozambican children that finds an associated between AggR and AAF/V fimbriae and Clade B isolates, predominating unexpectedly over Clade C. This counters the global trend, and so is of interest, as well as the region: there are really few samples from this geographic region, so that aspect alone is worth noting. Generally the study is good, though I have comments below on the nomenclature used, the stats in Table 1. A phylogeny would definitely enhance it to identify the fimH27 source. The major consideration is the genome assembly methods with the CLC workbench: no detail on the methods used is given, which undermines confidence in these results. More detail is below.

Comments:

- use the conventional Clade A/B/C nomenclature as well as of fimH41 / fimH22 / fimH30 - H27 is usually associated with subclade B0 (see Ludden et al 2019 https://doi.org/10.1101/814731, Ben Zakour et al 2016, Kallonen et al 2017, Stoesser et al 2016) or more likely just Clade B (Decano et al 2019 https://www.nature.com/articles/s41598-019-54004-5). The O sertoypes seem quite surprising in the context of previous work for ST131.

- Just FYI I believe the only other ST131 known to be from the Africa region are SRR1186472 and SRR1191664 (SRA accessions) from 2009 from Tanzania (many other related fimH27 samples do not have geographic information). Many others from outside Africa are available.

- Table 1 - are the 42 (approx) statistical tests applied and associated p values corrected for multiple testing? Some of the numbers in this table dom't add up - eg for genes aggR and aap the % of EAEC out of 44 is the same (41 and 41) but the % values are not. Later, no numbers are given for fimH and partially for agg4A. Please double-check. In addition, it would be easier for the reader IMHO if you split the table into two parts denoting the virulence factors associated with EAEC and those with the others: it is not easy to examine for ones in the latter category.

- This paper and inference of the ancestry of these isolates would be enhanced by a phylogeny of the core genome SNPs. Eg use Gubbins/ClonalFrameML to exclude recombinant tracts, then RAxML to construct the tree visualised with iTOL or FigTree. This would answer if they are the same outbreak, independent introductions from

- For the genome sequencing, much more detail is needed:

* How many reads were generated per sample? What were the mean and SD of their lengths, and likewise of their insert sizes?

* What QC (eg Fastp, etc) was done on the reads? Did you do base correction? (eg with BayesHammer/SPAdes/Pilon) (see Alikhan et al 2018 for an equivalent alternative approach)

* What were the assembly N50s, distribution of contig size lengths and how do you know the assembly process worked well? It is quite possible that some non-detected genes are a consequence of poorly assembled regions. Use Quast to look at the N50s, numbers of predicted genes/ORFs and numbers of contigs with mis-assemblies. Example standards as per Alikhan et al 2018 are assembly lengths of 3.7 to 6.4 Mb with less than 800 contigs and under 5% low-quality sites.

* How did you measure the assembly success say compared to the best available assembler Unicycler? Can you show that CLC generates assemblies not markedly inferior to this?

* What E. coli reference genome was used during the assembly process for scaffolding? (eg SSPACE, Mauve, etc)

* Did you annotate the genomes? (eg Prokka)

Reviewer #2: General comments:

This is an interesting, novel and clearly written manuscript. Results are described in a logical and straightforward manner. The discussion provides a balanced assessment of the author’s findings. In my view, one of the limitations of the study is the lack of information about the HIV infection in the population studied, considering that is performed in one of the regions with highest sero-prevalence of HIV in the world. HIV infections and immunosuppressive status generated by this infection could be a risk factor (maybe the more important) to develop an infection by these E. coli strains in the population studied.

Although this paper is focused on molecular epidemiology and virulence, a brief paragraph describing a bit more about antimicrobial resistance of the isolates studied and molecular mechanisms of resistance will bring even more interest to the manuscript. Authors, for instance says “our strains were susceptible to third generation cephalosporines and fluoroquinolones.” But, what about other antimicrobial families? Did any strain meet the MDR criteria? Line 332 says “WGS also identified genes conferring resistance towards three or more groups of antibiotics”, however this information does not indicate that they are MDR isolates.

CART analysis is interesting however it doesn´t take into account important variables of poor outcome such as antimicrobial resistance, adequacy of empiric and targeted treatments, HIV… This could be briefly discussed.

Could the authors classify the ST1313 isolates studied according to virotype classification (A to F)?

Specific comments:

Line 75, change symptoms by diseases.

Line 334: Change beta-lactamase by beta-lactams. Extend information about the antimicrobial encoding-genes found.

Table 3: Detail how the incidence rate was calculated.

Figure 2: Images have poor resolution

Line 420-421: Change “extended spectrum of β-lactamase” by broad spectrum β-lactams and “cephalosporines” by cephalosporins.

Reviewer #3: The paper of Mandomando and colleagues address the emergence of a new subclade of ST131 in a population of African bacteriemic children. Moreover, these isolates belonging to FimH27 contains both ExPEC and EAEC genes suggesting that this subclone may serve as a ‘melting pot’ for pathogroup conversion between EAEC and ExPEC. The paper is correctly written and may be of interest for readers however, I have some difficulties with some points. First, it is not clear what was done using PCR or serotyping and was done with WGS data analysis. Detection of pathotypes, virulence factors, and serotyping may be determined by in silico analysis. Secondly, among the 325 isolates collected for analysis, how were selected the 44 isolates for WGS? I understand that only EAEC isolates (identified with PCR methods) were sequenced. Is it true? How the authors justified this selection? Similarly, in the results section, the authors state that 66 isolates were serotyped, How the authors justified this selection?

Globally the collection analysis is not clear and the mode of isolate selection for further analysis should be more precisely described.

From my point of view, despite the novelty of the findings, the manuscript should be totally rewritten , I suggest the authors focused on description of emerging fimH27 ST131 subclade by comparing their own WGS data with ST131 WGS data available in NCBI

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Reviewer #2: No

Reviewer #3: No

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7 Apr 2020

Dear Dr. Mandomando,

We are pleased to inform you that your manuscript 'Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: emergence of new variant of fimH27subclone' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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Jorge Blanco, Ph. D.

Guest Editor

PLOS Neglected Tropical Diseases

Ana LTO Nascimento

Deputy Editor

PLOS Neglected Tropical Diseases

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

23 Apr 2020

Dear Dr. Mandomando,

We are delighted to inform you that your manuscript, "Escherichia coli ST131 clones harbouring AggR and AAF/V fimbriae causing bacteremia in Mozambican children: emergence of new variant of fimH27subclone," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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