Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh.

BACKGROUND: Burkholderia cepacia complex (Bcc) is a group of serious pathogens in cystic fibrosis patients and causes life threatening infections in immunocompromised patients. Species within the Bcc are widely distributed within the environment, can survive in the presence of disinfectants and antiseptics, and are inherently multidrug resistant (MDR).
METHODS: Dhaka Medical College Hospital (DMCH) patients with a B. cepacia positive blood culture between 20 October 2016 to 23rd September 2017 were considered as outbreak cases. Blood stream infections (BSIs) were detected using BacT/ALERT 3D at DMCH. B. cepacia was isolated on chromogenic UTI media followed by MALDI-TOF. Minimum inhibitory concentration (MIC) of clinically relevant antibiotics was determined by agar dilution. Whole genome sequencing was performed on an Illumina MiSeq platform. Patients' demographic and clinical data were collected. Patients' clinical history and genomic data of the outbreak strains were merged to investigate possible outbreaks. Ninety-one B. cepacia genomes were downloaded from 'Burkholderia Genome Database' and the genomic background of the global strains were compared with our outbreak strains.
RESULTS: Among 236 BSIs, 6.35% (15/236) were B. cepacia. Outbreak cases were confined to the burn critical care unit and, to a lesser extent, the paediatrics department. There was a continuum of overlapping cases at DMCH between 23 October 2016 to 30 August 2017. Core genome SNPs showed that the outbreak strains were confined to a single clade, corresponded to a common clone (ST1578). The strains were shown to be MDR and associated with a mortality of 31% excluding discharge against medical advice. MIC profiles of the strains suggested that antibiotics deployed as empirical therapy were invariably inappropriate. The genetic background of the outbreak strains was very similar; however, a few variations were found regarding the presence of virulence genes. Compared to global strains from the Burkholderia Genome Database, the Bangladeshi strains were genetically distinct.
CONCLUSIONS: Environmental surveillance is required to investigate the aetiology and mode of transmission of the B. cepacia outbreak. Systematic management of nosocomial outbreaks, particularly in resource limited regions, will mitigate transmission and will improve patients' outcomes.

Introduction

The genus Burkholderia incorporates Gram negative, catalase-producing, lactose-nonfermenting), obligately aerobic bacilli and encompasses the species of the (Bcc), , . and B. gladioli [1]. Bcc is composed of at least 20 different species, including B. cepacia, and [1,2]. Bcc was first described in mid-1980s, in cystic fibrosis (CF) patients as a cause of ‘Cepacia Syndrome’, characterized by lung function deterioration, bacteremia and death [3]. The members of Bcc are widely distributed in the environment, including water, soil, fruits and vegetables and can survive for prolong periods of time in moist environments, even in the presence of disinfectants and antiseptics. They are capable of colonizing fluids in the hospital such as irrigation solutions or intravenous fluids and, serve as potential source of nosocomial infections [4,5,6]. Bcc rarely cause diseases in healthy individuals and is mostly regarded as a serious pathogen in cystic fibrosis patients [7]. The pathogens are inherently multidrug resistant (MDR) and highly transmissible by direct contact [7-9].

Regardless of geographical location nosocomial outbreaks attributed by Bcc are frequent. Recent outbreaks of Bcc bacteraemia from India, Germany and USA, have been reported particularly in the immunocompromised patients [6, 10–12]. Bcc outbreaks are always linked to hospital environment and/or contaminated medical devices, intravenous fluids or antiseptic solutions [4-6,10-13]. We describe an outbreak of B. cepacia bacteraemia in burn’s critical care units of Dhaka Medical College Hospital (DMCH) analysed by whole genome sequencing. The population structure and genetic relationship of B. cepacia isolated in this study were compared with 91 global B. cepacia deposited previously in the ‘Burkholderia Genome Database’ [14].

Methods

Ethical statement

Ethical consent was given by the Ethical Review Committee (ERC) of Dhaka Medical College Hospital (DMCH) prior to the start of the study and in accordance with the Helsinki Declaration [Memo no: MEU-DMC/ECC/2017/122] [15]. Required written consent was taken from all the participants for this study.

Hospital setting and outbreak case definition

We carried out this study between 20 October 2016 and 23rd September 2017 in DMCH which is the largest public hospital in Bangladesh. A total of 527 blood culture samples received by the clinical microbiology laboratory were included in this study. Detailed microbiological analysis of isolates, including Whole Genome Sequencing (WGS), performed at Cardiff University, were undertaken retrospectively. Patients with a B. cepacia positive blood culture were included as outbreak cases. Patients’ demographic (name, age, sex, locality and socio-economic condition) and clinical data (clinical symptoms or reason for hospitalization, ward, type of infection, admission date, sample collection date, outcome and antimicrobial therapy) were collected. Clinical data was collected following positive culture results from the local laboratory. Additional clinical follow up information was not available in the current study, except for outcome (discharge, death, discharge against medical advice {DAMA}). The study outline is described in Fig 1.

Fig 1

Study flowchart.

Purification and screening of phenotypic resistance to the isolates of interest

Blood stream infections were detected using BacT/ALERT 3D (bioMerieux, North Carolina, USA) at DMCH. The isolates were sub-cultured onto chromogenic UTI agar (Liofilchem, Roseto, Italy) and the species were identified by Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometry (MS) (Bruker Daltonics, Bremen, Germany). Minimum inhibitory concentrations (MIC) to clinically relevant antimicrobials (amoxicillin-clavulanate, piperacillin-tazobactam, ceftriaxone, ceftazidime, cefotaxime, cefepime, imipenem, meropenem, ciprofloxacin, levofloxacin, amikacin, gentamicin, sulfamethoxazole-trimethoprim, fosfomycin, tigecycline and colistin) were determined by agar dilution and interpreted according to Clinical and Laboratory Standards Institute (CLSI) breakpoints [16]. MIC determination was carried out in triplicate.

Illumina MiSeq sequencing

WGS was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA). DNA libraries were prepared for paired end sequencing (2x301 cycles) using Nextera XT v2. Genomic DNA was extracted from overnight culture using the QIAcube (Qiagen, Hilden, Germany), and resulting gDNA was quantified using the Qubit 3.0 (Thermos Fisher Scientific, Waltham, USA). Quality control of raw reads included fastqc (v0.11.2) and adaptor trimming was performed using Trimgalore (v0.4.3). Reads were assembled into contigs using the de novo assembler SPAdes (v3.9.0) (.fasta) and were aligned to the original fastq reads using Burrows-Wheeler aligner (BWA) (v0.7.15). Any error was corrected using Pilon (v1.2). Assembly metrics were evaluated using Quast (v2.1). The de novo assembly produced multiple contigs (145–330), with a mean GC content of 50% and mean coverage 12X, annotated with Prokka (v1.12). Resistance genes were retrieved from Comprehensive Antibiotic Resistance Database (CARD) [17]. Mutations in ampD were mapped to ampD (BCAL3430) of B. cenocepacia strain J2315 which possesses a ceftazidime MIC of 2 μg/ml to evaluate the putative cause of ß-lactam resistance [14,18]. Virulence factors (VFs) were evaluated by Virulence Factor Database (VFDB) [19].

Multi locus sequence typing (MLST)

B. cepacia (n = 15) isolated in this study was submitted to pubMLST to assign as sequence types (STs) based on 7 loci [20]. Global B. cepacia (n = 91) genomes were downloaded from ‘Burkholderia Genome Database’ of which 82 strains could not be matched with previously described STs [14]. We also submitted the 82 genomes from database to pubMLST [20]. A minimum spanning phylogenetic tree was constructed with 106 B. cepacia genome using Ridom SeqSphere+ (Ridom GmbH, Münster, Germany) based on 7 B. cepacia MLST genes.

Core genome phylogenetic analysis

We mapped single nucleotide polymorphisms (SNPs) of global B. cepacia from database (n = 91) and our outbreak strains (n = 15) with the first isolated strain in our study, dm93 (used as reference genome) using Snippy (3.2) [21]. SNPs alignemnt was performed to build a high-resolution phylogeny. We constructed a maximum likelihood (ML) tree using FastTree (2.1.3). We employed Interactive Tree of Life (iTOL) to visualize the tree [22].

Investigating possible outbreaks

Data extracted from patients’ clinical history and WGS data of the outbreak strains were evaluated to investigate possible outbreaks. Likely epidemiological links on transmission (patient to patient connectivity) was predicted if there is overlapping of hospital stay among the outbreak cases (Fig 2). Detailed epidemiological information and subsequent infection control measures were not available as the microbiological information was only available retrospectively. Infection Prevention and Control (IPC) measures were not implemented at the time due to the limited and untimely microbiological information at the DMCH.

Fig 2

Length of hospital stay of the patients with B. cepacia bacteraemia.

Accession numbers

WGS data of all B. cepacia in this study were deposited in the National Centre for Biotechnology Information under accession numbers stated below.

Accession                      Organism

SNSF00000000        Burkholderia cepacia b219

SNSG00000000        Burkholderia cepacia b212

SNSH00000000        Burkholderia cepacia b163

SNSI00000000        Burkholderia cepacia b124

SNSJ00000000        Burkholderia cepacia b111

SNSK00000000        Burkholderia cepacia b101

SNSL00000000        Burkholderia cepacia b100

SNSM00000000        Burkholderia cepacia b98

SNSN00000000        Burkholderia cepacia b84

SNSO00000000        Burkholderia cepacia b64

SNSP00000000        Burkholderia cepacia b19

SNSQ00000000        Burkholderia cepacia b13

SNSR00000000        Burkholderia cepacia b6

SNSS00000000        Burkholderia cepacia dm93

SSHL00000000        Burkholderia cepacia b99

Results

Study population and overview of B. cepacia cases

Among 527 blood culture specimens, 45% (236/527) were culture positive and 3% (15/527) were B. cepacia bacteraemia (Fig 1). Of the 15 B. cepacia infected patients, 7 (46.7%) were male and 8 (53.3%) were female; the mean (±SD) age was 14 years (±12.57). All the patients in this cohort belonged to a low socio-economic group (lower middle to below the national poverty level). The mean (±SD) hospital stay of B. cepacia cases was 43.9 days (±27.1). Antibiotic usage among the cases with B. cepacia septicaemia was: ceftriaxone, 80.0%; amikacin, 53.3%; levofloxacin, 33.3%; ceftazidime, 20%; meropenem, 13.3%, colistin, 13.3%; flucloxacillin, 6.7%; azithromycin, 6.7%; gentamicin, 6.7%; clindamycin, 6.7%; cefuroxime, 6.7% (Table 1). Two of the B. cepacia cases took discharge against medical advice (DAMA). Not including DAMA, the mortality rate was 31% (4/13) (Fig 2; Table 1). Patient characteristics are illustrated in Table 1. The first case was admitted to paediatrics department of DMCH on the 23rd of October 2016. According to hospital records, the patient was a suspected case of sepsis, and blood was sent for culture and sensitivity on the 2nd of November 2016 which was positive for B. cepacia. The rest of the cases were confined to the burn HDU or the burn paediatric HDU or the burn ICU (Fig 2; Table 1). Excluding DAMA (5/135), in the burn unit, the mortality rate for patients with other bacterial sepsis was higher (60.17%, 71/118) compared to B. cepacia sepsis (33.33%, 4/12) (p<0.1).

Table 1

Clinical characteristics of patients infected with B. cepacia.

Strain ID	Cases	Ward	Sample	Agea	Sex	SEC	DOA	DOSC	DOD	THS	Underlying disease	Antibiotic history	Outcome
dm93	Case 1	Pae	Blood	2.5	M	BPL	23.10.16	02.11.16	7.11.16	16	Unknownb	CRO, GEN	Discharge
b19	Case 2	Burn HDU	Blood	30	F	BPL	25.10.16	22.11.16	21.1.17	58	35% FB	CRO	DAMA
b06	Case 3	Burn HDU	Blood	22	M	Poor	10.11.16	22.11.16	30.11.16	21	35% FB	CFU, CLN	Discharge
b13	Case 4	Burn HDU	Blood	45	F	BPL	13.11.16	22.11.16	26.11.16	14	25% FB	AK, CAZ	Died
b64	Case 5	Burn Pae. HDU	Blood	3.5	F	Poor	14.12.16	02.01.17	2.2.17	51	15% FB	AZ, CL, CRO	Discharge
b84	Case 6	Burn HDU	Blood	15	M	BPL	14.01.17	26.01.17	23.3.17	69	40% EB	AK, CAZ, MEM	Discharge
b98	Case 7	Burn ICU	Blood	5	F	BPL	04.02.17	09.02.17	10.2.17	7	45% FB with II	AK, CRO, FLU	Died
b99	Case 8	Burn ICU	Blood	10	F	LM	03.02.17	09.02.17	09.02.17	7	30% MBc	CRO, LEVO	DAMA
b100	Case 9	Burn Pae. HDU	Blood	2.5	F	LM	28.01.17	09.02.17	8.4.17	71	22% SB	AK, CRO, LEVO	Discharge
b101	Case 10	Burn Pae. HDU	Blood	7	M	BPL	03.02.17	09.02.17	17.2.17	15	43% FB	AK, CRO	Died
b111	Case 11	Burn HDU	Blood	19	M	Poor	16.01.17	23.02.17	7.4.17	82	30% FB	AK, CRO, MEM	Discharge
b124	Case 12	Burn Pae. HDU	Blood	3.5	F	BPL	25.02.17	05.03.17	23.4.17	58	30% FB	CAZ, CL, CRO	Died
b163	Case 13	Burn ICU	Blood	1.5	M	Poor	10.4.17	17.4.17	26.5.17	46	40% SB	CRO, LEVO	Discharge
b212	Case 14	Burn HDU	Blood	23	F	Poor	20.5.17	31.5.17	22.6.17	34	35% FB	AK, CRO, LEVO	Discharge
b219	Case 15	Burn HDU	Blood	20	M	Poor	15.6.17	19.6.17	30.8.17	76	35% EB with II	AK, CRO, LEVO	Discharge

Cases were delineated according to date of isolation of B. cepacia chronologically. ICU, intensive care unit; HDU, high dependency unit, M, male; F, female; SEC, socio-economic condition; BPL, below poverty level, LM, lower middle; DOA, date of admission; DOSC, date of sample collection; DOD, date of discharge/DAMA/death; THS, total hospital stay; Pae, paediatrics; FB, flame burn, EB, electric burn; II, inhalation injury; MB, mixed bun; SC, scald burn; DAMA, discharge against medical advice. AK, amikacin; AZ, azithromycin; CAZ, ceftazidime; CFU, cefuroxime; CL, colistin; CLN, clindamycin; CRO, ceftriaxone; FLU, flucloxacillin; GEN, gentamicin; LEVO, levofloxacin; MEM, meropenem

aAge are given in years

bUnderlying disease was not available in hospital record; however, this case was clinically suspected as sepsis and therefore, blood was referred for culture

cCombination of chemical and flame burn

Clonal relationship with global B. cepacia

B. cepacia isolated in this study were assigned as a single novel ST, ST1578 (Fig 3). All strains (n = 106) were grouped into 9 clusters (Fig 3). The Bangladeshi strains shared ST clusters with strains from Australia, USA, South Korea, Thailand, UK and Malaysia. Interestingly, STs differed according to geographical area (Fig 3). Core genome alignment also suggested that the Bangladeshi B. cepacia are genetically closer to environmental strains of Australian origin rather than human strains isolated from cystic fibrosis patients in the UK (LMG16656.fsa nt), Thailand (LO6.fasta), and other Asian strains (Fig 4).

Fig 3

Minimum spanning tree of B. cepacia by MLST type.

Each node within the tree represents a single ST. The size of the nodes is proportional to the number of isolates represented by corresponding node. Selected nodes are labelled with corresponding STs, and number of isolates represented. All global strains including Bangladeshi outbreak strains mentioned in this diagram were assigned as corresponding STs in this study except ST10, ST44, ST807 and ST810.

Fig 4

A maximum likelihood tree of B. cepacia by core genome SNPs with epidemiological data and antimicrobial resistance genes.

Country of origin is represented by specific colour of node. Node level are highlighted according to source of sample. Global strains are stated with specific codes. Original strain IDs with corresponding codes are compiled with supplementary data (S1 Data). ARG, antimicrobial resistance genes; U, unknown.

Outbreak investigation

We documented this outbreak between October 2016 and August 2017 at DMCH (Fig 2). All outbreak cases had clinical signs of sepsis at the time of sample collection and patients in burn critical units were undergoing artificial ventilation and had central venous catheter lines and urinary catheters at the time of diagnosis. There was a continuous overlapping of patients at DMCH during the outbreak period (Table 1; Fig 2). Core genome SNPs showed that the outbreak strains were confined to a single clade (Fig 4; S1 Data), corresponded to a common clone (ST1578) (Fig 3).

Background on resistance and virulence

MICs were performed in triplicate and all repetitions were within one-dilution. There are no MIC breakpoints for B. cepacia recommended by EUCAST [23]. According to CLSI breakpoints, 20% of B. cepacia (in this study) were resistant to ceftazidime and 93.33% intermediate resistant to levofloxacin. All the outbreak strains had high MIC value for amoxicillin-clavulanate, amikacin, gentamicin, fosfomycin, trimethoprim-sulfamethoxazole and colistin (Table 2). All were sensitive to meropenem (Table 2) [16]. MIC value of relevant antimicrobials are stated in Table 2. According to their MIC profiles, antibiotics deployed as empirical therapy to treat outbreak cases were invariably deemed to be inappropriate (Table 1). The antimicrobial resistance genes identified in the outbreak strains were identical (Fig 4; S1 Data). Likewise, ampD for all strains was homogenous, however, compared to ampD (BCAL3430) of B. cenocepacia strain J2315, we found 10 substitutions of amino acid in ampD (S1 Fig). The outbreak strains were shown to possess more resistant genes than other global strains (Fig 4; S1 Data).

Table 2

MIC value of B. cepacia in this study against relevant antibiotics (n = 15).

Strain ID	AUGa	Pip-Taza	CROa	CAZa	CTXa	CEFa	IMPa	MEMa	CIPa	LEVOa	AKa	GENa	Fosa	SXT-TRMa	Tigea	Cla
dm93	>256	4	8	4	16	8	8	4	2	4	64	>256	>256	32	16	>256
b19	>256	8	16	8	32	16	8	4	1	4	256	>256	>256	64	4	>256
b06	>256	8	16	8	32	16	8	4	1	4	256	>256	>256	32	16	>256
b13	>256	8	16	8	32	16	8	4	2	4	256	>256	>256	32	16	>256
b64	>256	2	4	2	8	8	8	2	2	4	64	>256	>256	64	8	>256
b84	>256	8	16	4	16	16	8	4	2	4	256	>256	>256	64	16	>256
b98	>256	2	4	2	8	8	8	2	2	4	64	>256	>256	64	8	>256
b99	>256	16	32	8	32	32	8	4	2	4	256	>256	>256	32	4	>256
b100	>256	8	16	4	32	16	8	4	2	4	256	>256	>256	64	8	>256
b101	>256	2	4	2	8	8	8	2	2	4	64	>256	>256	32	8	>256
b111	>256	2	4	2	8	8	8	2	1	2	256	>256	>256	64	4	>256
b124	>256	2	16	8	16	16	8	4	2	4	256	>256	>256	64	16	>256
b163	>256	16	32	16	32	8	8	4	2	4	128	>256	>256	32	16	>256
b212	>256	8	16	16	16	16	8	4	2	4	128	>256	>256	64	16	>256
b219	>256	16	32	32	32	32	8	4	2	4	64	>256	>256	64	16	>256

aMIC values are indicated by mg/l

MIC values were determined in triplicate. AK, amikacin; AUG, amoxicillin-clavulanate; Pip-Taz, piperacillin-tazobactam; Cl, colistin; CRO, ceftriaxone; CAZ, ceftazidime; CTX, cefotaxime; CEF, cefepime; Fos, fosfomycin; GEN, gentamicin; IMP, imipenem; MEM, meropenem; CIP, ciprofloxacin; LEVO, levofloxacin; SXT-TRM, trimethoprim-sulfamethoxazole; Tige, tigecycline

The Bangladeshi outbreak strains shared some common virulence genes with the global strains (bimA, boaAB, pilABCDNOQRSTV, gmhA, manC, wcbABCDEFGHIJKLMNOPQRST, wzm/wzt2, cheABDRWYZ, flgABCDEFGHIJKLMN, fliACDEFGHIJKLMNOPQRS, motAB, tsr, bspI2/bspI3, bspR2/bspR3/bspR4/bspR5, pmlI/bspI1, pmlR/bspR1, bapABC, basJ, bicAP, bipBCD, bopACE, bprABCDPQ, bsaKLMNOPQRSTUVXYZ, orgAB, spaP, gene for T6SS), although two of the global strains (MSMB1338WGS.fsa_nt and DWS16B-4.fsa_nt) were negative for all genes from VFDB (Fig 5; S1 Data). There was variation in the presence of virulence genes between our clinical outbreak strains and previous clinical B. cepacia (LMG16656.fsa nt and LO6.fasta) isolated from CF patients (Fig 5; S1 Data).

Fig 5

Heatmap showing the presence of virulence genes in B. cepacia.

Global strains are stated with specific codes. Original strain IDs with corresponding codes are compiled with supplementary data (S1 Data). No VF was found in MSMB1338WGS.fsa_nt and DWS16B-4.fsa_nt. Apart from this two strains, other strains were shown to be positive for common virulence genes (bimA, boaAB, pilABCDNOQRSTV, gmhA, manC, wcbABCDEFGHIJKLMNOPQRST, wzm/wzt2, cheABDRWYZ, flgABCDEFGHIJKLMN, fliACDEFGHIJKLMNOPQRS, motAB, tsr, bspI2/bspI3, bspR2/bspR3/bspR4/bspR5, pmlI/bspI1, pmlR/bspR1, bapABC, basJ, bicAP, bipBCD, bopACE, bprABCDPQ, bsaKLMNOPQRSTUVXYZ, orgAB, spaP, gene for T6SS). These common VFs were not included in heatmap. Violet indicates the presence and grey indicates the absence of VF in the respective strain. Virulence genes in operon (orfHM, aaiAB, vscNR, ureBG, vasAG, farAB, lpxAC, vapA1/vapA, rmlAC, katAG, syrCD, ifgBGH, algCUW, rmlABC, clpB.clpV/clpV1, adeFGH, pvdAFH, phzB2/D2/E2/F2, hemBCEL, flhABCDFG, pchABCDEFGHIR) are represented by the intensity of violet—the increase in intensity is proportional to number of genes.

Discussion

Although B. cepacia is considered almost exclusively a pathogen for CF patients [3,7], our study reported B. cepacia bacteraemia predominantly from burns patients. B. cepacia are considered opportunistic human pathogens and can be transmitted to patients via environmental contamination or person-to-person contact [4-6,8,9]. Burns patients are generally more susceptible to infection due to impaired immune function. Risk factors for sepsis in burns include >20% of total body surface area (TBSA), inhalation injury, delayed burn wound excision, increased length of hospital stay, use of artificial medical devices and ICU admission [24-28]. Patients in burn ICUs are more vulnerable to septicaemia than general ICU patients [29]. Our findings demonstrate an outbreak of bacteraemia in DMCH caused by a single clone of B. cepacia ST1578, mostly confined to patients admitted in burn critical care units (Fig 2). The outbreak cases had a mean hospital stay of 43.9 days and straddled each other implying transmission via direct patient contact and/or patient to hospital environment and vice versa. Clinically, this outbreak was associated with a mortality rate of 31% (Fig 2; Table 1).

Previous studies show that burn sepsis accounts for 50–60% of deaths in burn patients [30,31]. In this study, mortality due to burn sepsis with bacteria other than B. cepacia (60.17%) was significantly higher than B. cepacia sepsis (33.33%) (p<0.1). A limitation of the current study was the lacked sufficient clinical information to access and to investigate why the mortality rate was significantly lower for B. cepacia cases. At the time of enrolment many of these patients were on “inadequate” antibiotics based on our susceptibility data. However, there are limitations to the interpretation of AST results for B. cepacia and we lack data on whether antibiotics were changed later during hospital stay. It is possible there were other factors favouring survival in the group infected with B. cepacia, including possibly relatively lower pathogenicity of B. cepacia as a cause of bacteraemia. B. cepacia has been described as a cause of pseudo-outbreaks [32]. Although this possibility should be considered here the timing of infections suggest this was not the case, and a persistent environmental source of cross contamination on the burn unit was a more likely cause. Unsurprisingly, radical infection control programs can mitigate the spread of infections and improve patient outcomes in burn units [33]. No IPC intervention was undertaken here because the outbreak was identified retrospectively.

MDR Gram-negative bacteria infections have become a serious challenge in health care settings as a result of both intrinsic and acquired resistance mechanisms, limiting therapeutic options [8]. B. cepacia is of concern due to their intrinsic resistance to clinically relevant antibiotics such as aminoglycosides and polymyxins [34]. In this study, all B. cepacia showed very high MIC breakpoints to amikacin and gentamicin mediated by resistance genes ant, aph and aadA2 and/or overexpression of efflux pump such as ArmA, CoeA (Fig 4; S1 Data) [34-36]. Burkholderia spp. are typically resistance to colistin due to a unique intrinsic amino arabinose biosynthesis operon [33]. Genome sequencing also identified resistance genes such as adeF, qacH, tetC and sul1 (Fig 4; S1 Data) which contribute to phenotypic resistance of B. cepacia (Table 2). Mutations in ampD are associated with the upregulation of ß-lactams degrading enzymes, PenB and AmpC. This mechanism has been found to be one of the causes of ß-lactam resistance in B. cepacia [18,34]. Mutations in ampD were identical in all B. cepacia analysed in our study and all the isolates had high MIC value for amoxicillin-clavulanate; however, MICs for piperacillin-tazobactam and cephalosporins were variable (Table 2). Perhaps enzymatic degradation was mainly responsible for putative ß-lactam resistance in this study although we did not evaluate the expression of ß-lactamase such as PenB, AmpC or PenA in relation to AmpD mutations [18,34]. Although the empirical antibiotics were shown to be ineffective (Table 1), the relationship between patients’ morbidity/mortality and antibiotics prescription cannot be fully explored as datasets are incomplete.

Virulence of B. cepacia is typically related to adhesins, invasins, intracellular pathogenicity, antiphagocytic factors, secretory and signalling systems [19,37]. A set of virulence genes in relation to all steps in pathogenesis were identified in Bangladeshi outbreak strains B. cepacia (Fig 5; S1 Data). However, whether other virulence determinants are associated with the ability to cause bacteraemia, and the role of patient factors, could not be determined by this current study.

B. cepacia identified in this study belonged to a novel clonal type ST1578 (Fig 3). The genetic background of the outbreak strains was very similar; however, a few variations were found regarding the presence of virulence genes (Fig 5; S1 Data). Compared to global strains from the database, resistance genes for aminoglycosides (ant(2'')-Ia, aph(6)-Id, aph(3'')-Ib, aadA2), fluoroquinolones (qacH), tetracycline (tet(C)) and sulphonamide (sul1) were found only among the strains isolated in this study (Fig 4; S1 Data). Virulence gene, aai was absent in our outbreak strain which was common in both human strains, LMG16656.fsa nt and LO6.fasta, (from Burkholderia Genome Database), but the overall virulence pattern of the outbreak strains was most similar to LMG16656.fsa nt (human strain isolated from the UK) than LO6.fasta (human strain isolated from Thailand). Compared to B. cepacia LO6.fasta, flagellar protein (lfg, lfh, lfi), secretory system (clpV1, vsc, iagB, spaR), regulatory protein (prrA) were absent in the Bangladeshi strains (Fig 5; S1 Data). Thus, it can be inferred that the Bangladeshi outbreak strains identified in this study are genetically distinct from global strains retrieved from the database.

Although we did not fully evaluate the epidemiological link between patients and the environment, epidemiological and molecular data suggest that the outbreak clone was circulating within DMCH over a protracted period (Fig 2). It is disconcerting that antibiotic pressure might have enhanced the elevation in antibiotic resistance during the outbreak [35,36]. Identification of environmental sources of the outbreak followed by patient management can reduce the risk of infections in vulnerable populations [4-6,10-13,29].

Substitutions of amino acid in ampD compared to ampD (BCAL3430) of B. cenocepacia strain J2315.

Substitutions are underlined by red.

(TIF)

Click here for additional data file.

Fig 4, Table 2, Fig 5.

(XLSX)

Click here for additional data file.

16 Nov 2019

Dear Dr Refath Farzana:

Thank you very much for submitting your manuscript "Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh" (#PNTD-D-19-01418) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the manuscript as it currently stands. These issues must be addressed before we would be willing to consider a revised version of your study. We cannot, of course, promise publication at that time.

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Deputy Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

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

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: This paper describes the use of whole genome sequencing to describe the phylogeny of B. cepacia isolates recovered over an 11 month period from in-patients hospitalised in burn critical care units in a large public hospital in Bangladesh. The experimental approaches used to examine the isolates are appropriate; however, the techniques used are not novel, although the application of WGS to B. cepacia isolates is still relatively uncommon. They are in the whole reasonably well presented although would benefit from clarification of some details (some suggestions below but not exhaustive) and provision of a rationale for the study should be provided.

Areas for improvement include clarification if it is indeed a retrospective study (if not then rationale for undertaking study-i.e. clinical suspicion, increase in incidence over baseline, etc.), if the 527 blood culture samples were all that were collected during the 11 month study period (and if these included the entire hospital’s workload or just those from the specific burn critical care units), if only 15 B. cepacia blood culture positive samples were identified during this period (again hospital wide versus specific units), if other B. cepacia isolates were recovered contemporaneously, including non-bloodstream infection related and environmental isolates.

Minor points:

p5, ln10: Suggest re naming to “Culture, identification and antimicrobial susceptibility testing of B. cepacia isolates”

p5, ln108: include phrase re absence of EUCAST guidelines here rather than in Results section

p5, ln110: Suggest re write section to improve flow: i.e. provide detail re method of genomic DNA purification, include if manufacturer’s instructions were followed, usual nomenclature is 2 x 300 cycles, remove detail re actual GC content & depth of coverage to Results but include genome coverage calculation method, identification of resistance genes (as distinct from ‘retrieval’), if contigs were reordered against ATCC reference genome prior to annotation.

P6, ln123 section: Clarify if MLSTs for the study isolates were determined from WGS reads or following traditional 7-locus amplification with Sanger sequencing.

P6, ln131 section: Describe process of generating reference genome assembly from dm93. Was a cgMLST approach considered for phylogenetic analysis and a wgMLST approach for virulence and resistance genotype analysis?

P6, ln134: change ‘aligemnt’ to ‘alignment’

No detail or references to statistical methods used are included.

Reviewer #3: This is an outbreak, however, it's not defined in the methodology as to what attempts were made to find out the source, if any.

The same needs to be clarified.

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

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: The results describe the findings of an epidemiological investigation of 15 B. cepacia isolates recovered over an 11 period from in-patients hospitalised in burn critical care units in a large public hospital in Bangladesh. Clinical backdrop along with antimicrobial susceptibility and molecular characteristics of the isolates are presented. The authors use WGS data of 91 B. cepacia strains taken from a publically available genome database to demonstrate the distinct clonal nature of their isolates. It is evident from this manuscript that the authors have performed a level of background clinical investigation and sequence analysis of their isolates to contextualise and fully document the outbreak, although much more could be presented on WGS data analysis. Furthermore, the results are not presented clearly and as a consequence they are difficult to follow (some suggestions for improvement provided, but again not an exhaustive list).

Minor points:

Include a section to detail WGS QC statistics, depth of coverage, %GC (from Methods), N50, etc. & also any statistics relating to the 91 global genomes if these differ from in-house isolate genomes.

P9, ln197: Suggest this section be moved to before p9, ln178 section.

All of Table 1 is not visible, probably because of conversion to pdf

Fig 1: One blood culture positive case is not accounted for.

P7, ln144 section and Fig 2: It would be useful if some idea of bed capacity, occupancy rates and medical staff:patient ratios were available, as well as consideration of other underlying health conditions/risk factors (extent of burns, etc.) to better understand the mortality rate. Perhaps also an analysis of antibiotic treatment in relation to organism susceptibility could be considered and correlated to duration of infections and case outcome. These may well though form part of Table 1 and just be not visible to reviewer.

P9, ln178 section: Although this reviewer has little background in B. cepacia phylogeny, analysis of MLST and cgSNP is useful although in this instance the inclusion of only a few other human derived isolates does perhaps skew the results (possible that differences are distinct clade-associated), however perhaps WGS data from other human derived isolates are not available in publically accessible databases such as NCBI or BIGSdb. Could it be that two distinct clones exist within the Bangladeshi isolates (one recovered during the weeks around Feb 1st 2017), with evidence of microevolution within each over the course of the study? Were topologies of MLST and cgSNP trees compared?

Fig 3: No scale is provided to assist with interpretation.

Fig 4: While very impressive looking, it is difficult to discern the node colour detail. No scale is provided to assist with interpretation.

Table 2: The data could be better represented using S/R interpretations.

P10, ln205 section and Table 2: Differences (greater than the tolerance of a doubling dilution on either side) in MIC values between strains are evident despite all isolates exhibiting the same resistance gene profiles; did the authors probe this further with respect to genotypes correlating with other antimicrobial resistance related mechanisms?

P11, ln225: Improve details of differences in VF repertoire among Bangladeshi isolates; comment on potential significance of absence of lfiP, htpB, virB1/virB4 and phzB2/D2/E2/F2 from some Bangladeshi isolates; occurrence on plasmids, etc.

P11, ln230: suggest rewording to “no virulence genes were identified in two of the global strains (MSMB1338WGS.fas_nt and DWS16B-4.fsa_nt) using VFDB.”

Fig 5: No scale is provided to assist with interpretation. Also, it is difficult to read the virulence genes

Reviewer #3: This is an outbreak, however, it's not defined in the methodology as to what attempts were made to find out the source, if any.

It should have been followed by the results of the attempts to find out the source.

It needs to be stated.

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

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 authors correctly concluded that the Bangladeshi B. cepacia isolates were from a single clone belonging to ST1578 and suggest that its continuance over the 11 month period was most likely related to transmission events with the burn critical care units. However, using the larger cgSNP dataset indicates that perhaps two similar clones (albeit of the same ST) were actually present; although without a scale on Figs 4 and 5 and also the inclusion of mainly environmental strains in the analysis with only a few other human-derived strains external to this time period and locale, it is difficult to be certain.

Many important points are raised in the Discussion that unfortunately could not be adequately addressed due to lack of available information. In addition it is unclear as to how the conclusion relating to the “elevation of antibiotic resistance during the outbreak” was supported. Furthermore, many of the conclusions stated in the abstract, author summary and cover letter are not demonstrated in the Results and not developed in the Discussion.

Reviewer #3: The authors have concluded on the note of contamination, which hasn't been actually searched for in the whole manuscript.

It needs to be changed as the conceptualization of the manuscript isn't proper and it doesn't as such match with the title of the manuscript also.

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Some suggestions to improve the flow and readability of the text are included in the previous sections but these are not exhaustive.

Reviewer #3: Minor revision

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: (No Response)

Reviewer #2: The manuscript entitled “Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh” by Farzana et al details an investigation of 15 cases of B. cepacia bloodstream infection over an 11 month period in burns critical care units in Dhaka Medical College Hospital, Bangladesh. The authors examine if the cases are due to the spread of a single strain of B. cepacia. From using whole genome sequencing the authors establish that the 15 isolates were genetically very homogenous (compared to 91 other global B. cepacia, mainly from environmental sources) and harboured common antimicrobial resistance genes and virulence factors (distinct from the global B. cepacia) and conclude that the Bangladeshi isolates belonged to a single clone. A clone that was allowed to persist in the burn critical care units, due to inadequate antibiotic treatment and a lack of appropriate infection prevention and control measures.

The topic of this manuscript is interesting considering the global struggle with multi-drug resistant organisms and will be of interest to the readers of the journal. It is one of the first papers that documents the use of whole genome sequencing to understand a clinical problem involving B. cepacia. It also highlights some of the challenges faced by the state health system in Bangladesh including overcrowding, inconsistent antimicrobial prescribing policies and variable infection control and prevention policies, all of which contributed to the unrestricted continuation of this strain over the 11-month study period. However, the manuscript would benefit from extensive revision and also a more in depth and appropriate analysis of the data with a better presentation and detailing of the results and discussion with appropriate references.

Reviewer #3: 1. In methods, it's not defined as to which breakpoints were used for MIC though described under Results in line 206 onwards, however, the year of CLSI Manual is not mentioned.

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

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Submitted filename: Comments.docx

Click here for additional data file.

3 Dec 2019

Submitted filename: rebuttal.docx

Click here for additional data file.

13 Jan 2020

Dear Dr. Refath Farzana:

Thank you very much for submitting your manuscript "Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh" (PNTD-D-19-01418R1) for review by PLOS Neglected Tropical Diseases. Your manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important topic but identified some aspects of the manuscript that should be improved.

We therefore ask you to modify the manuscript according to the review recommendations before we can consider your manuscript for acceptance. Your revisions should address the specific points made by each reviewer.

In addition, when you are ready to resubmit, please be prepared to provide the following:

(1) A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript.

(2) Two versions of the manuscript: one with either highlights or tracked changes denoting where the text has been changed (uploaded as a "Revised Article with Changes Highlighted" file ); the other a clean version (uploaded as the article file).

(3) If available, a striking still image (a new image if one is available or an existing one from within your manuscript). If your manuscript is accepted for publication, this image may be featured on our website. Images should ideally be high resolution, eye-catching, single panel images; where one is available, please use 'add file' at the time of resubmission and select 'striking image' as the file type.

Please provide a short caption, including credits, uploaded as a separate "Other" file. If your image is from someone other than yourself, please ensure that the artist has read and agreed to the terms and conditions of the Creative Commons Attribution License at http://journals.plos.org/plosntds/s/content-license (NOTE: we cannot publish copyrighted images).

(4) Appropriate Figure Files

Please remove all name and figure # text from your figure files upon submitting your revision. Please also take this time to check that your figures are of high resolution, which will improve both the editorial review process and help expedite your manuscript's publication should it be accepted. Please note that figures must have been originally created at 300dpi or higher. Do not manually increase the resolution of your files. For instructions on how to properly obtain high quality images, please review our Figure Guidelines, with examples at: http://journals.plos.org/plosntds/s/figures

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. 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 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 hope to receive your revised manuscript by Mar 13 2020 11:59PM. If you anticipate any delay in its return, we ask that you let us know the expected resubmission date by replying to this email.

To submit your revised files, please log in to https://www.editorialmanager.com/pntd/

If you have any questions or concerns while you make these revisions, please let us know.

Sincerely,

Ruifu Yang

Deputy Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

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

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: More detailed information may be need for the definition of the outbreak. Considering the DMCH is a tertiary care hospital, the Infection Control Division would have taken actions for better disinfection and clinical investigation, rather than observing the hospital stay over 11 months. Such a long time for a response to nosocomial infection emergency. Authors should provide more detailed information about clinical investigation.

Reviewer #3: Acceptable.

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

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: Burkholderia cepacia sepsis outbreak among human is rare and neglected in clinic. The results basically match the analysis plan and the evidence is sufficient for the definition of the outbreak from the perspective of molecular epidemiological technology. The results are clearly demonstrated and the figures are of sufficient quality. If possibel, more background information of clinical investigation is better to predict the epidemiological links between cases. The gap between resistance gene prediction and clinical antimicrobial phenotype is often obvious.

Reviewer #3: -Are the results clearly and completely presented?

No.

If we look at the table 1, an important finding is there that those patients who were given empirical antibiotic either levofloxacin or meropenem were discharged (though no. is small) and none of them died. Authors need to look at the susceptibility of these antibiotics in the given patient. Accordingly, this information may be included in the manuscript.

Another observation is that in case 1 & 3, despite on other antibiotics, patients get discharged. Did these patients have clinical signs of sepsis or do these cases fall under pseudobacteremia for which B. cepacia complex is well known?

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

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: Generally, BC is not common pathogen in burn care unit. This is an out-of-control emergency of nosocomial infection. So donnot mention too much abouth the universality of BC infection in burn ICU, but take a deep research about the definition of the criminal bacteria and the links between cases.

Reviewer #3: -Are the conclusions supported by the data presented?

No.

Conclusions: Environmental contamination is a potential source of outbreak in a hospital setting with very poor infection control policies.

The manuscript lacks the data to support this conclusion. Only the isolates from burn ward have been collected without even mentioning about the possible source.

Despite pointing out earlier, it has still not been addressed as a limitation in the manuscript.

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

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: Accept after minor revision in Investigating possible outbreaks in Methods section.

Reviewer #3: None

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

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: This study is interesting for the Burkholderia spp. researchers and useful for the doctors in clinic in Burkholderia spp.-endemic regions. This paper is easily-understood, precise, and suitable for the aim of this maggazine, neglected diseases.

Reviewer #3: None

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

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.

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Reviewer #1: No

Reviewer #3: No

31 Jan 2020

Submitted filename: rebuttal-31-01-20.docx

Click here for additional data file.

10 Feb 2020

Dear DR. Refath Farzana,

Thank you very much for submitting your manuscript "Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh" 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. The reviewers appreciated the attention to an important topic. Based on the reviews, we are likely to accept this manuscript for publication, providing that you modify the manuscript according to the review recommendations.

Please prepare and submit your revised manuscript within 30 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to all review comments, and a description of the changes you have made in the 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

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

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Sincerely,

Ruifu Yang

Deputy Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

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

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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. 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.

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5 Mar 2020

Dear Dr. Farzana,

We are pleased to inform you that your manuscript 'Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

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

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

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

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Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Ruifu Yang

Deputy Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

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

2 Apr 2020

Dear Dr. Farzana,

We are delighted to inform you that your manuscript, "Molecular and epidemiological analysis of a Burkholderia cepacia sepsis outbreak from a tertiary care hospital in Bangladesh," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

We have now passed your article onto the PLOS Production Department who will complete the rest of the publication process. All authors will receive a confirmation email upon publication.

The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any scientific or type-setting errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Note: Proofs for Front Matter articles (Editorial, Viewpoint, Symposium, Review, etc...) are generated on a different schedule and may not be made available as quickly.

Soon after your final files are uploaded, the early version of your manuscript will be published online unless you opted out of this process. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.

Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Serap Aksoy

Editor-in-Chief

PLOS Neglected Tropical Diseases

Shaden Kamhawi

Editor-in-Chief

PLOS Neglected Tropical Diseases