Genomic Insights Into the Mechanism of Carbapenem Resistance Dissemination in Enterobacterales From a Tertiary Public Heath Setting in South Asia.

SUMMARY: 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone. BACKGROUND: Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017. METHODS: We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales (CSE). RESULTS: 10.6% of all 1831 patients with a clinical specimen collected had CRE. In-hospital 30-day mortality was significantly higher with CRE [50/180 (27.8%)] than CSE [42/312 (13.5%)] (P = .001); however, for bloodstream infections, this was nonsignificant. Of 643 Enterobacterales isolated, 210 were CRE; blaNDM was present in 180 isolates, blaOXA-232 in 26, blaOXA-181 in 24, and blaKPC-2 in 5. Despite this, ceftriaxone was the most commonly prescribed empirical antibiotic and only 27% of patients were prescribed at least 1 antibiotic to which their infecting pathogen was susceptible. Significant risk factors for CRE isolation included burns unit and intensive care unit admission, and prior exposure to levofloxacin, amikacin, clindamycin, and meropenem. Escherichia coli ST167 was the dominant CRE clone. Clustering suggested clonal transmission of Klebsiella pneumoniae ST15 and the MDR hypervirulent clone, ST23. The major trajectories involved in horizontal gene transfer were IncFII and IncX3, IS26, and Tn3. CONCLUSIONS: This is the largest study from an SA public hospital combining outcome, microbiology, and genomics. The findings indicate the urgent implementation of targeted diagnostics, appropriate antibiotic use, and infection-control interventions in SA public institutions.

BKC-2, a New BKC Variant Detected in MCR-9.1-Producing Enterobacter hormaechei subsp. xiangfangensis.

We characterized a multidrug-resistant (MDR) Enterobacter spp. isolate highlighting the genetic aspects of the antimicrobial resistance genes. An Enterobacter spp. isolate (Ec61) was recovered in 2014 from a transtracheal aspirate sample from a patient admitted to a Brazilian tertiary hospital and submitted to further microbiological and genomic characterization. Ec61 was identified as Enterobacter hormaechei subsp. xiangfangensis strain ST451, showing an MDR profile and the presence of genes codifying the new ß-lactamase variants BKC-2 and ACT-84 and the mobile colistin resistance gene mcr-9.1.

Outbreak of Hypervirulent Multidrug-resistant Klebsiella variicola Causing High Mortality in Neonates in Bangladesh.

We report a clonal outbreak of multidrug-resistant (MDR) Klebsiella variicola (sequence type [ST] 771) in a Bangladeshi neonatal unit from October 2016 to January 2017, associated with high mortality (54.5%). During the outbreak, K. variicola ST771 acquired an MDR plasmid harboring blaNDM-1, linked to high exposure to ceftriaxone and amikacin.

Antimicrobial susceptibility pattern of extended spectrum beta-lactamase producing gram-negative bacteria isolated from wound and urine in a tertiary care hospital, Dhaka City, Bangladesh.

From a total of 320 bacterial samples from wound swab and urine 169 (53%) gram-negative bacteria were isolated, of which 42 (25%) extended-spectrum beta-lactamase (ESBL) producers were detected by double-disk synergy test. ESBL producers were significantly more resistant against amoxiclav, Co-trimoxazole, ciprofloxacin, amikacin and gentamicin than non-ESBL producers. Among the 42 ESBL producers, 76% were positive for blaCTX-M and 43% were positive for blaOXA, with blaCTX-M predominantly (97%) observed in E. coli and blaOXA predominantly (80%) in Pseudomonas spp. Class 1 integron was found in 75% of blaCTX-M positive and 56% of blaOXA positive strains. Combinations of ESBL genes and class 1 integron were observed in 29 (69%) of the ESBL producers. The findings of this study infer that CTX-M and OXA producers are emerging in Bangladesh and we report the presence of blaOXA for the first time in Bangladesh.

International manufacturing and trade in colistin, its implications in colistin resistance and One Health global policies: a microbiological, economic, and anthropological study.

BACKGROUND: The emergence of colistin-resistant Enterobacterales is a global public health concern, yet colistin is still widely used in animals that are used for food as treatment, metaphylaxis, prophylaxis, and growth promotion. Herein, we investigate the effect of colistin-resistant Enterobacterales in Pakistan, global trade of colistin, colistin use at the farm level, and relevant socioeconomic factors. METHODS: We conducted a microbiological, economic, and anthropological study of colistin-resistant Escherichia coli in humans, animals, and the environment and international trade and knowledge of colistin in Pakistan, Bangladesh, Nigeria, China, India, and Viet Nam. We collected backyard poultry cloacal swabs, commercial broiler cloacal swabs, cattle and buffalo rectal swabs, human rectal swabs, wild bird droppings, cattle and buffalo meat, sewage water, poultry flies, chicken meat, and canal water from 131 sites across Faisalabad, Pakistan, to be tested for mcr-1-positive and mcr-3-positive Escherichia coli. We recruited new patients admitted to Allied Hospital, Faisalabad, Pakistan, with abdominal pain and diarrhoea for rectal swabs. Patients with dysentery and those who were already on antibiotic treatment were excluded. Data for colistin trade between 2017 and 2020, including importation, manufacturing, and usage, were accessed from online databases and government sources in Pakistan, Bangladesh, and Nigeria. We recruited participants from poultry farms and veterinary drug stores in Pakistan and Nigeria to be interviewed using a structured questionnaire. International manufacturing, import, and export data; value analysis; and trade routes of colistin pharmaceutical raw material (PRM), feed additive, and finished pharmaceutical products (FPPs) were accessed from 2017-21 export data sets. FINDINGS: We collected 1131 samples between May 12, 2018, and July 1, 2019: backyard poultry cloacal swabs (n=100), commercial broiler cloacal swabs (n=102), cattle and buffalo rectal swabs (n=188), human rectal swabs (n=200), wild bird droppings (n=100), cattle and buffalo meat (n=100), sewage water (n=90), poultry flies (n=100), chicken meat (n=100), and canal water (n=51). We recruited 200 inpatients at Allied Hospital, Faisalabad, Pakistan, between Nov 15, 2018, and Dec 14, 2018, for rectal swabs. We recruited 21 participants between Jan 1, 2020, and Dec 31, 2020, from poultry farms and drug stores in Pakistan and Nigeria to be interviewed. 75 (7%) of 1131 samples contained mcr-1-positive E coli, including wild bird droppings (25 [25%] of 100), commercial broiler cloacal swabs (17 [17%] of 100), backyard poultry cloacal swabs (one [1%] of 100), chicken meat (13 [13%] of 100), cattle and buffalo meat (two [2%] of 100), poultry flies (eight [8%] of 100), sewage water (six [7%] of 90), and human rectal swabs (three [2%] of 200). During 2017-20, Pakistan imported 275·5 tonnes (68·9 tonnes per year, 95% CI 41·2-96·6) of colistin as PRM, all sourced from China, 701·9 tonnes (175·5 tonnes per year, 140·9-210·1) of colistin as feed additives from China and Viet Nam, and 63·0 tonnes (15·8 tonnes per year, 10·4-21·1) of colistin as FPPs from various countries in Asia and Europe. For Bangladesh and Nigeria, colistin PRM and FPPs were imported from China and Europe. Colistin knowledge and usage practices in Pakistan and Nigeria were unsatisfactory in terms of understanding of the effects on human medicine and usage other than for treatment purposes. China is the major manufacturer of PRM and feed additive colistin and exported a total of 2664·8 tonnes (666·2 tonnes per year, 95% CI 262·1 to 1070·2) of PRM and 2570·2 tonnes (642·6 tonnes per year, -89·4 to 1374·5) of feed additive in 1330 shipments during 2018-21 to 21 countries. INTERPRETATION: Regardless of 193 countries signing the UN agreement to tackle antimicrobial resistance, trading of colistin as PRM, FPPs, and feed additive or growth promoter in low-income and middle-income countries continues unabated. Robust national and international laws are urgently required to mitigate the international trade of this antimicrobial listed on WHO Critically Important Antimicrobials for Human Medicine. FUNDING: Pakistan Agricultural Research Council and INEOS Oxford Institute for Antimicrobial Research TRANSLATION: For the Urdu translation of the abstract see Supplementary Materials section.

Molecular and genetic characterization of emerging carbapenemase-producing Acinetobacter baumannii strains from patients and hospital environments in Bangladesh.

Background: Carbapenemase-producing multidrug-resistant (MDR) Acinetobacter baumannii is a global health care problem. MDR A. baumannii has emerged as an important nosocomial pathogen, costing many lives worldwide including Bangladesh. Aim: To investigate the detailed molecular epidemiology of carbapenem-resistant A. baumannii (CRAB) both from patients and the hospital environment, to shed light on genetic characteristics and transmission dynamics. Methods: A set of 49 clinical A. baumannii strains collected during early 2015 was received from the clinical microbiology laboratory of Dhaka Medical College Hospital (DMCH) in Bangladesh. Additionaly, 100 environmental samples were also collected from the hospital surfaces of Dhaka Medical College Hospital and analyzed for carbapenamase-producing A. baumannii. CRAB were identified by culture on selective plates, biochemical testing and MALDI-TOF. All isolates were characterized by susceptibility testing, realtime-PCRs, conventional PCR, MLST and sequencing. Findings: Clinical A. baumannii were resistant to ciprofloxacin (100%), imipenem (91.8%), meropenem (91.8%), gentamicin (91.8%), amikacin (87.7%), and trimethoprim-sulfamethoxazole (61.2%). The majority (59%) of the isolates were MDR. All environmental A. baumannii (n=10) were resistant to imipenem, meropenem, gentamicin, amikacin, and ciprofloxacin. Strains carried the following antibiotic resistant genes; bla OXA-23, bla OXA-58, bla PER-7, qnrB1, qnrC1, aac(6')1b-cr and armA. A total of 36 different clones were identified by rep-PCR and common clonal clusters were found both in patients and hospital environments. MLST analysis revealed different sequence types (ST2, ST10, ST149, ST575, ST1063 and ST1065). In clinical and environmental settings. A. baumannii ST2 dominated in both clinical and environmental settings. Both clinical and environmental A. baumannii strains with known STs carried several biofilm-related genes; bap, csuE, and pgaB. Conclusion: Widespread dissemination of MDR A. baumannii in the DMC hospital of Bangladesh is a serious problem.

Effects of antibiotic resistance, drug target attainment, bacterial pathogenicity and virulence, and antibiotic access and affordability on outcomes in neonatal sepsis: an international microbiology and drug evaluation prospective substudy (BARNARDS).

BACKGROUND: Sepsis is a major contributor to neonatal mortality, particularly in low-income and middle-income countries (LMICs). WHO advocates ampicillin-gentamicin as first-line therapy for the management of neonatal sepsis. In the BARNARDS observational cohort study of neonatal sepsis and antimicrobial resistance in LMICs, common sepsis pathogens were characterised via whole genome sequencing (WGS) and antimicrobial resistance profiles. In this substudy of BARNARDS, we aimed to assess the use and efficacy of empirical antibiotic therapies commonly used in LMICs for neonatal sepsis. METHODS: In BARNARDS, consenting mother-neonates aged 0-60 days dyads were enrolled on delivery or neonatal presentation with suspected sepsis at 12 BARNARDS clinical sites in Bangladesh, Ethiopia, India, Pakistan, Nigeria, Rwanda, and South Africa. Stillborn babies were excluded from the study. Blood samples were collected from neonates presenting with clinical signs of sepsis, and WGS and minimum inhibitory concentrations for antibiotic treatment were determined for bacterial isolates from culture-confirmed sepsis. Neonatal outcome data were collected following enrolment until 60 days of life. Antibiotic usage and neonatal outcome data were assessed. Survival analyses were adjusted to take into account potential clinical confounding variables related to the birth and pathogen. Additionally, resistance profiles, pharmacokinetic-pharmacodynamic probability of target attainment, and frequency of resistance (ie, resistance defined by in-vitro growth of isolates when challenged by antibiotics) were assessed. Questionnaires on health structures and antibiotic costs evaluated accessibility and affordability. FINDINGS: Between Nov 12, 2015, and Feb 1, 2018, 36 285 neonates were enrolled into the main BARNARDS study, of whom 9874 had clinically diagnosed sepsis and 5749 had available antibiotic data. The four most commonly prescribed antibiotic combinations given to 4451 neonates (77·42%) of 5749 were ampicillin-gentamicin, ceftazidime-amikacin, piperacillin-tazobactam-amikacin, and amoxicillin clavulanate-amikacin. This dataset assessed 476 prescriptions for 442 neonates treated with one of these antibiotic combinations with WGS data (all BARNARDS countries were represented in this subset except India). Multiple pathogens were isolated, totalling 457 isolates. Reported mortality was lower for neonates treated with ceftazidime-amikacin than for neonates treated with ampicillin-gentamicin (hazard ratio [adjusted for clinical variables considered potential confounders to outcomes] 0·32, 95% CI 0·14-0·72; p=0·0060). Of 390 Gram-negative isolates, 379 (97·2%) were resistant to ampicillin and 274 (70·3%) were resistant to gentamicin. Susceptibility of Gram-negative isolates to at least one antibiotic in a treatment combination was noted in 111 (28·5%) to ampicillin-gentamicin; 286 (73·3%) to amoxicillin clavulanate-amikacin; 301 (77·2%) to ceftazidime-amikacin; and 312 (80·0%) to piperacillin-tazobactam-amikacin. A probability of target attainment of 80% or more was noted in 26 neonates (33·7% [SD 0·59]) of 78 with ampicillin-gentamicin; 15 (68·0% [3·84]) of 27 with amoxicillin clavulanate-amikacin; 93 (92·7% [0·24]) of 109 with ceftazidime-amikacin; and 70 (85·3% [0·47]) of 76 with piperacillin-tazobactam-amikacin. However, antibiotic and country effects could not be distinguished. Frequency of resistance was recorded most frequently with fosfomycin (in 78 isolates [68·4%] of 114), followed by colistin (55 isolates [57·3%] of 96), and gentamicin (62 isolates [53·0%] of 117). Sites in six of the seven countries (excluding South Africa) stated that the cost of antibiotics would influence treatment of neonatal sepsis. INTERPRETATION: Our data raise questions about the empirical use of combined ampicillin-gentamicin for neonatal sepsis in LMICs because of its high resistance and high rates of frequency of resistance and low probability of target attainment. Accessibility and affordability need to be considered when advocating antibiotic treatments with variance in economic health structures across LMICs. FUNDING: The Bill & Melinda Gates Foundation.

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.

Emergence of Mobile Colistin Resistance (mcr-8) in a Highly Successful Klebsiella pneumoniae Sequence Type 15 Clone from Clinical Infections in Bangladesh.

The emergence of mobilized colistin resistance genes (mcr) has become a serious concern in clinical practice, compromising treatment options for life-threatening infections. In this study, colistin-resistant Klebsiella pneumoniae harboring mcr-8.1 was recovered from infected patients in the largest public hospital of Bangladesh, with a prevalence of 0.3% (3/1,097). We found mcr-8.1 in an identical highly stable multidrug-resistant IncFIB(pQil) plasmid of ∼113 kb, which belonged to an epidemiologically successful K. pneumoniae clone, ST15. The resistance mechanism was proven to be horizontally transferable, which incurred a fitness cost to the host. The core genome phylogeny suggested the clonal spread of mcr-8.1 in a Bangladeshi hospital. Core genome single-nucleotide polymorphisms among the mcr-8.1-positive K. pneumoniae isolates ranged from 23 to 110. It has been hypothesized that mcr-8.1 was inserted into IncFIB(pQil) with preexisting resistance loci, blaTEM-1b and blaCTX-M-15, by IS903B Coincidentally, all resistance determinants in the plasmid [mcr-8.1, ampC, sul2, 1d-APH(6), APH(3'')-Ib, blaTEM-1b, blaCTX-M-15] were bracketed by IS903B, demonstrating the possibility of intra- and interspecies and intra- and intergenus transposition of entire resistance loci. This is the first report of an mcr-like mechanism from human infections in Bangladesh. However, given the acquisition of mcr-8.1 by a sable conjugative plasmid in a successful high-risk clone of K. pneumoniae ST15, there is a serious risk of dissemination of mcr-8.1 in Bangladesh from 2017 onwards.IMPORTANCE There is a marked paucity in our understanding of the epidemiology of colistin-resistant bacterial pathogens in South Asia. A report by Davies and Walsh (Lancet Infect Dis 18:256-257, https://doi.org/10.1016/S1473-3099(18)30072-0, 2018) suggests the export of colistin from China to India, Vietnam, and South Korea in 2016 was approximately 1,000 tons and mainly used as a poultry feed additive. A few reports forecast that the prevalence of mcr in humans and livestock will increase in South Asia. Given the high prevalence of blaCTX-M-15 and blaNDM in India, Bangladesh, and Pakistan, colistin has become the invariable option for the management of serious infections, leading to the emergence of mcr-like mechanisms in South Asia. Systematic scrutiny of the prevalence and transmission of mcr variants in South Asia is vital to understanding the drivers of mcr genes and to initiate interventions to overcome colistin resistance.

Emergence of mcr-1 mediated colistin resistant Escherichia coli from a hospitalized patient in Bangladesh.

INTRODUCTION: The emergence of plasmid mediated mcr in bacteria has become global public health threat. Herein, we report a mcr-1 positive E. coli in normal human flora from a patient admitted in Dhaka Medical College Hospital (DMCH). METHODOLOGY: In total, 700 non-duplicate rectal swabs were collected from DMCH during 13th May to 12th June 2018. E. coli from rectal swabs were isolated on chromogenic UTI media containing vancomycin 10mg/l (Liofilchem, Italy) and confirmed by MALDI-TOF. Minimum inhibitory concentrations (MIC) were determined by agar dilution and interpreted according to EUCAST breakpoints. Genomic analysis of mcr positive E. coli (MCRPEC) was performed by Illumina MiSeq sequencing and pulsed field gel electrophoresis (PFGE) using S1 nuclease DNA digests and blamcr-1 probing. Transferability of blamcr-1 were determined by conjugation assays. RESULTS: We found one MCRPEC from 700 rectal swab screening which was isolated from the rectal swab culture of a 17-year boy who was admitted to the burns ICU, DMCH with 53% flame burn involving much of the trunk and face. Genome sequencing revealed that mcr-1 was present on an IncH12 plasmid of 257,243 bp and flanked by ISApaI1. The colistin resistance can be transferred to the recipient Klebsiella varricola with a frequency of 8.3 × 10-5. Transconjugants were more resistant to colistin than donor (MIC 32 µg/mL). CONCLUSIONS: This is the first human associated mcr in Bangladesh. These data indicate the need for a systematic "one health" surveillance in the country.

Isolation and molecular characterization of New Delhi metallo-beta-lactamase-1 producing superbug in Bangladesh.

INTRODUCTION: New Delhi metallo-beta-lactamse-1 (NDM-1) producing superbugs create a global public health problem because of their resistance to most antibiotics. This study was conducted to determine the presence of MBL producers, including NDM-1 producers, in Bangladesh, along with the antimicrobial resistance patterns of these organisms. METHODOLOGY: Thirty-five isolates resistant to imipenem by disk diffusion technique were investigated for MBL production. Minimum inhibitory concentration (MIC) of imipenem was determined by agar dilution method. MBL producers were phenotypically detected by double disk synergy test and combined disk assay. Gene encoding blaIMP-1, blaIMP-2, blaVIM-1, blaVIM-2, blaNDM-1 and class 1 integron was identified by PCR. RESULTS: Thirty-one (88.57%) MBL producers were detected by PCR, 24 (68.57%) by double disk synergy test, and 30 (85.71%) by combined disk assay. Eight (22.86%) were positive for blaNDM-1, 13 (37.15%) for blaVIM-1, 21 (60.00%) for blaVIM-2, 18 (51.43%) for blaIMP-1, and 9 (25.71%) for blaIMP-2. More than one blaMBL was present in 23 (65.71%) of the isolates. MIC of imipenem of MBL producers ranged from ≥256 µg/ml to ≤8 µg/ml. All the NDM-1 producing isolates carried class 1 integron. NDM-1 producers were 100% resistant to amoxicillin, cephradine, cefuroxime, ceftazidime, cefotaxime, ceftriaxone, aztreonam, gentamicin and piperacillin, 87.5% to amikacin, 75% to ciprofloxacin, and 62.5% to co-trimoxazole and the combination of tazobactam and piperacillin. All were sensitive to colistin. CONCLUSION: The results of this study provide insight into the presence of blaMBL, including blaNDM-1, in Bangladesh. Urgent epidemiological monitoring of MBL producers in Bangladesh may combat their rapid dissemination.