Whole genome-based genetic insights of blaNDM producing clinical E. coli isolates in hospital settings of Pakistan.

Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo ß-lactamase NDM have been isolated from two hospitals in Faisalabad, Pakistan. These E. coli strains were characterized by MALDI-TOF, PCR, antimicrobial susceptibility testing, XbaI and S1 nuclease pulsed-field gel electrophoresis (PFGE), conjugation assay, DNA hybridization, whole genome sequencing, bioinformatic analysis, and Galleria mellonella experiments. Thirty-four blaNDM producing E. coli strains were identified among 52 nonduplicate carbapenem-resistant strains. More than 90% of the isolates were found to be multidrug resistant by antimicrobial susceptibility testing. S1 PFGE confirmed the presence of blaNDM gene on plasmids ranging from 40 kbps to 250 kbps, and conjugation assays demonstrated transfer frequencies of blaNDM harboring plasmids ranging from 1.59 × 10-1 to 6.46 × 10-8 per donor. Whole genome sequencing analysis revealed blaNDM-5 as the prominent NDM subtype with the highest prevalence of blaOXA-1, blaCTX-M-15, aadA2, aac(6')-Ib-cr, and tet(A) associated resistant determinants. E. coli sequence types: ST405, ST361, and ST167 were prominent, and plasmid Inc types: FII, FIA, FIB, FIC, X3, R, and Y, were observed among all isolates. The genetic environment of blaNDM region on IncF plasmids included partial ISAba125, the bleomycin ble gene, and a class I integron. The virulence genes terC, traT, gad, fyuA, irp2, capU, and sitA were frequently observed, and G. mellonella experiments showed that virulence correlated with the number of virulence determinants. A strong infection control management in the hospital is necessary to check the emergence of carbapenem resistance in Gram-negative bacteria.IMPORTANCEWe describe a detailed analysis of highly resistant clinical E. coli isolates from two tertiary care centers in Pakistan including carbapenem resistance as well as common co-resistance mechanisms. South Asia has a huge problem with highly resistant E. coli. However, we find that though these isolates are very difficult to treat they are of low virulence. Thus the Western world has an increasing problem with virulent E. coli that are mostly of low antibiotic resistance, whereas, South Asia has an increasing problem with highly resistant E. coli that are of low virulence potential. These observations allow us to start to devise methodologies to limit both virulence and resistance and combat problems in developing nations as well as the Western world.

Diversity of lytic bacteriophages against XDR Klebsiella pneumoniae sequence type 16 recovered from sewage samples in different parts of the world.

Bacteriophages (phages) are viruses considered to be natural bacterial predators and widely detected in aquatic environments. Sewage samples are an important source of phage isolation since high density and diversity of bacterial cells are present, due to human, animal and household fluids. This study aims to investigate and characterise phages against an extremely drug-resistant (XDR) lineage, Klebsiella pneumoniae ST16, using sewage samples from different parts of the World. Sewage samples from Brazil, Bangladesh, Saudi Arabia, Thailand and the United Kingdom were collected and used to investigate phages against ten K. pneumoniae ST16 (hosts) recovered from infection sites. The phages were microbiological and genetically characterised by double-agar overlay (DLA), transmission electron microscopy and Illumina WGS. The host range against K. pneumoniae belonging to different sequence types was evaluated at different temperatures by spot test. Further phage characterisation, such as efficiency of plating, optimal phage temperature, and pH/temperature susceptibility, were conducted. Fourteen lytic phages were isolated, belonging to Autographiviridae, Ackermannviridae, Demerecviridae, Drexlerviridae, and Myoviridae families, from Brazil, Bangladesh, Saudi Arabia and Thailand and demonstrated a great genetic diversity. The viruses had good activity against our collection of clinical K. pneumoniae ST16 at room temperature and 37 °C, but also against other important Klebsiella clones such as ST11, ST15, and ST258. Temperature assays showed lytic activity in different temperatures, except for PWKp18 which only had activity at room temperature. Phages were stable between pH 5 and 10 with minor changes in phage activity, and 70 °C was the temperature able to kill all phages in this study. Using sewage from different parts of the World allowed us to have a set of highly efficient phages against an K. pneumoniae ST16 that can be used in the future to develop new tools to combat infections in humans or animals caused by this pathogen.

Effective phage cocktail to combat the rising incidence of extensively drug-resistant Klebsiella pneumoniae sequence type 16.

Bacteriophages are the most abundant organisms on Earth. As there are few effective treatment options against some pathogens, the interest in the bacteriophage control of multi-drug-resistant bacterial pathogens is escalating, especially for Klebsiella pneumoniae. This study aimed to develop a phage-based solution to the rising incidence of extensively drug-resistant clinical Klebsiella pneumoniae sequence type (ST16) infections starting from a set of phages recently characterized against this lineage. A phage-cocktail (Katrice-16) composed of eight lytic phages was characterized for potential use in humans. In vitro and in vivo broth inhibition and Galleria mellonella rescue assays were used to demonstrate the efficacy of this approach using a collection of 56 strains of K. pneumoniae ST16, with distinct genetic backgrounds that were collected from clinical infections from four disparate nations. Additionally, Katrice-16 anti-biofilm activity, synergism with meropenem, and activity in human body fluids were also assessed. Katrice-16 was highly active in vitro against our K. pneumoniae ST16 collection (AUC% median = 86.48%; Q1 = 83.8%; Q2 = 96.85%; Q3 = 98.85%). It additionally demonstrated excellent in vivo activity in G. mellonella rescue assays, even with larvae infected by isolates that exhibited moderate in vitro inhibition. We measured significant anti-biofilm activity over 12 h (p = .0113) and synergic activity with meropenem. In addition, we also demonstrate that Katrice-16 maintained high activity in human body fluids. Our results indicate that our cocktail will likely be an effective solution for human infections with this increasingly prevalent and often highly resistant bacterial clone.

Complete Genome Sequence of the Virulent Klebsiella pneumoniae Phage Geezett Infecting Multidrug-Resistant Clinical Strains.

Geezett was isolated from hospital sewage in Hangzhou, China, and exhibits lytic activity against clinical isolates of the nosocomial pathogen Klebsiella pneumoniae. The bacteriophage is a myovirus and has a double-stranded DNA (dsDNA) genome 50,707 bp long, containing 79 open reading frames (ORFs).

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.

Clinical and Molecular Description of a High-Copy IncQ1 KPC-2 Plasmid Harbored by the International ST15 Klebsiella pneumoniae Clone.

This study provides the genomic characterization and clinical description of bloodstream infections (BSI) cases due to ST15 KPC-2 producer Klebsiella pneumoniae Six KPC-K. pneumoniae isolates were recovered in 2015 in a tertiary Brazilian hospital and were analyzed by whole-genome sequencing (WGS) (Illumina MiSeq short reads). Of these, two isolates were further analyzed by Nanopore MinION sequencing, allowing complete chromosome and plasmid circularization (hybrid assembly), using Unicycler software. The clinical analysis showed that the 30-day overall mortality for these BSI cases was high (83%). The isolates exhibited meropenem resistance (MICs, 32 to 128 mg/liter), with 3/6 isolates resistant to polymyxin B. The conjugative properties of the blaKPC-2 plasmid and its copy number were assessed by standard conjugation experiments and sequence copy number analysis. We identified in all six isolates a small (8.3-kb), high-copy-number (20 copies/cell) non-self-conjugative IncQ plasmid harboring blaKPC-2 in a non-Tn4401 transposon. This plasmid backbone was previously reported to harbor blaKPC-2 only in Brazil, and it could be comobilized at a high frequency (10-4) into Escherichia coli J53 and into several high-risk K. pneumoniae clones (ST258, ST15, and ST101) by a common IncL/M helper plasmid, suggesting the potential of international spread. This study thus identified the international K. pneumoniae ST15 clone as a carrier of blaKPC-2 in a high-copy-number IncQ1 plasmid that is easily transmissible among other common Klebsiella strains. This finding is of concern since IncQ1 plasmids are efficient antimicrobial resistance determinant carriers across Gram-negative species. The spread of such carbapenemase-encoding IncQ1 plasmids should therefore be closely monitored.IMPORTANCE In many parts of the world, carbapenem resistance is a serious public health concern. In Brazil, carbapenem resistance in Enterobacterales is mostly driven by the dissemination of KPC-2-producing K. pneumoniae clones. Despite being endemic in this country, only a few reports providing both clinical and genomic data are available in Brazil, which limit the understanding of the real clinical impact caused by the dissemination of different clones carrying blaKPC-2 in Brazilian hospitals. Although several of these KPC-2-producer K. pneumoniae isolates belong to the clonal complex 258 and carry Tn4401 transposons located on large plasmids, a concomitant emergence and silent dissemination of small high-copy-number blaKPC-2 plasmids are of importance, as described in this study. Our data identify a small high-copy-number IncQ1 KPC plasmid, its clinical relevance, and the potential for conjugative transfer into several K. pneumoniae isolates, belonging to different international lineages, such as ST258, ST101, and ST15.

Clinical utilization of bacteriophages: a new perspective to combat the antimicrobial resistance in Brazil

ABSTRACT Due to the emergence of multi-drug resistant bacteria, and the evident limitation in therapeutic options, alternatives to combat bacterial infections have been sought. One of these is phage therapy, which is the use of bacterial viruses to kill pathogenic bacteria responsible for the infection. These viruses called bacteriophages are very abundant organisms in the world and are harmless to humans. There are several advantages in using phage therapy, especially against multi-drug resistant pathogens, which tend to be dominated by individual strains. The advantages include fewer collateral effects such as lower disturbance of gut microbiota and less antimicrobials consumption, which itself leads to reducing antibiotic resistance rates. Unfortunately, few clinical studies have been initiated in Brazil and this area is little explored in our country. This manuscript describes clinical evidence of successful phage utilization on pathogens considered a threat in Brazil, highlighting the benefits of a possible phage utilization as an important tool to combat antimicrobial resistance in our country.

Clinical utilization of bacteriophages: a new perspective to combat the antimicrobial resistance in Brazil.

Due to the emergence of multi-drug resistant bacteria, and the evident limitation in therapeutic options, alternatives to combat bacterial infections have been sought. One of these is phage therapy, which is the use of bacterial viruses to kill pathogenic bacteria responsible for the infection. These viruses called bacteriophages are very abundant organisms in the world and are harmless to humans. There are several advantages in using phage therapy, especially against multi-drug resistant pathogens, which tend to be dominated by individual strains. The advantages include fewer collateral effects such as lower disturbance of gut microbiota and less antimicrobials consumption, which itself leads to reducing antibiotic resistance rates. Unfortunately, few clinical studies have been initiated in Brazil and this area is little explored in our country. This manuscript describes clinical evidence of successful phage utilization on pathogens considered a threat in Brazil, highlighting the benefits of a possible phage utilization as an important tool to combat antimicrobial resistance in our country.

The present danger of New Delhi metallo-ß-lactamase: a threat to public health.

The evolution of antimicrobial-resistant Gram-negative pathogens is a substantial menace to public health sectors, notably in developing countries because of the scarcity of healthcare facilities. New Delhi metallo-ß-lactamase (NDM) is a potent ß-lactam enzyme able to hydrolyze several available antibiotics. NDM was identified from the clinical isolates of Klebsiella pneumoniae and Escherichia coli from a Swedish patient in New Delhi, India. This enzyme horizontally passed on to various Gram-negative bacteria developing resistance against a variety of antibiotics which cause treatment crucial. These bacteria increase fatality rates and play an integral role in the economic burden. The efficient management of NDM-producing isolates requires the coordination between each healthcare setting in a region. In this review, we present the prevalence of NDM in children, fatality and the economic burden of resistant bacteria, the clonal spread of NDM harboring bacteria and modern techniques for the detection of NDM producing pathogens.

Human carriage of cefotaxime-resistant Escherichia coli in North-East India: an analysis of STs and associated resistance mechanisms.

OBJECTIVES: To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India. METHODS: E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes. RESULTS AND CONCLUSIONS: Analysis of isolates collected without selection (n=33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15.

Extended-spectrum ß-lactamase-producing Escherichia coli in human-derived and foodchain-derived samples from England, Wales, and Scotland: an epidemiological surveillance and typing study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Escherichia coli isolates (ESBL-E coli) cause more than 5000 cases of bacteraemias annually in the UK. The contribution of the food chain to these infections is debated. We aimed to identify the most important reservoirs of ESBL-E coli that colonise and infect humans to identify strategic intervention points. METHODS: Sampling for ESBL-E coli was done between Aug 1, 2013, and Dec 15, 2014. We used selective media to seek ESBL-E coli in routinely submitted samples from human faeces, and prospectively collected samples from sewage, farm slurry, and retail foodstuffs in London, East Anglia, northwest England, Scotland, and Wales. We sequenced recovered isolates and compared these isolates with 293 bloodstream and 83 veterinary surveillance ESBL-E coli isolates from the same regions. FINDINGS: 2157 (11%) of 20 243 human faeces samples contained ESBL-E coli, including 678 (17%) of 3995 in London. ESBL-E coli also were frequent in sewage and retail chicken (104 [65%] of 159 meat samples), but were rare in other meats and absent from plant-based foods (0 of 400 fruit and vegetable samples). Sequence type (ST) 131 dominated among ESBL-E coli from human blood (188 [64%] of 293 isolates), faeces (128 [36%] of 360), and sewage (14 [22%] of 65) with STs 38 and 648 also widespread; CTX-M-15 was the predominant ESBL in these lineages (319 [77%] of 416). By contrast, STs 602, 23, and 117-mostly with CTX-M-1 ESBL-dominated among food and veterinary isolates (68 [31%] of 218), with only two ST131 organisms recovered. ST10 occurred in both animals and humans, being frequent in surveillance bovines (11 [22%] of 51 cattle) and representing 15 (4%) of 360 human faecal isolates (but only three [1%] of 293 from bacteraemias); however, both human and animal ST10 isolates were diverse in serotype. INTERPRETATION: Most human bacteraemias with ESBL-E coli in the UK involve internationally prevalent human-associated STs, particularly ST131; non-human reservoirs made little contribution to invasive human disease. Any interventions that seek to target food or livestock can affect the numbers of human infections caused by ESBL-E coli; prevention of the spread of resistant lineages among humans is more vital. FUNDING: NIHR Policy Research.

Dissemination of genetically diverse NDM-1, -5, -7 producing-Gram-negative pathogens isolated from pediatric patients in Pakistan.

Aim: To determine the prevalence of New Delhi metallo-ß-lactamase (NDM)-producing Gram-negative pathogens isolated from children's samples. Materials & methods: Carbapenem-resistant clinical isolates (n = 117) were confirmed by VITEK® 2 compact system, matrix-assisted laser desorption ionization-time of flight and multilocus sequence typing. MIC (µg/ml) of various antibiotics was determined by VITEK 2 compact system. Molecular characterization of the isolates was performed by PCR, DNA sequencing, PFGE and DNA hybridization. Results: Out of 117 carbapenemase producers, 37 (31.6%) and 29 (24.7%) were Klebsiella pneumoniae and Acinetobacter baumannii, respectively. 72 (61.5%) isolates were NDM positive and among these 60, 9 and 3 were NDM-1, -5 and -7, respectively. Majority of the NDM-producing K. pneumoniae belonged to ST11 and ST273 while most of the Escherichia coli belonged to ST405 and ST101. blaNDM were mainly located on 150kb plasmids. MIC displayed high resistance against ß-lactams drugs including carbapenems, and the most sensitive drugs were tigecycline and colistin. Conclusion: Dissemination of blaNDM-producing pathogens, particularly in children clinical settings, is a matter of great public health concern.

OXA-1 ß-lactamase and non-susceptibility to penicillin/ß-lactamase inhibitor combinations among ESBL-producing Escherichia coli.

Background: ESBL-producing Escherichia coli have expanded globally since the turn of the century and present a major public health issue. Their in vitro susceptibility to penicillin/inhibitor combinations is variable, and clinical use of these combinations against ESBL producers remains controversial. We hypothesized that this variability related to co-production of OXA-1 penicillinase. Methods: During a national study we collected 293 ESBL-producing E. coli from bacteraemias, determined MICs by BSAC agar dilution, and undertook genomic sequencing with Illumina methodology. Results: The collection was dominated by ST131 (n = 188 isolates, 64.2%) and blaCTX-M-15 (present in 229 isolates, 78.2%); over half the isolates (159/293, 54.3%) were ST131 with blaCTX-M-15. blaOXA-1 was found in 149 ESBL producers (50.9%) and blaTEM-1/191 in 137 (46.8%). Irrespective of whether all isolates were considered, or ST131 alone, there were strong associations (P < 0.001) between co-carriage of blaOXA-1 and reduced susceptibility to penicillin/inhibitor combinations, whereas there was no significant association with co-carriage of blaTEM-1/191. For piperacillin/tazobactam the modal MIC rose from 2 mg/L in the absence of blaOXA-1 to 8 or 16 mg/L in its presence; for co-amoxiclav the shift was smaller, from 4 or 8 to 16 mg/L, but crossed the breakpoint. blaOXA-1 was strongly associated with co-carriage also of aac(6')-Ib-cr, which compromises amikacin and tobramycin. Conclusions: Co-carriage of OXA-1, a penicillinase with weak affinity for inhibitors, is a major correlate of resistance to piperacillin/tazobactam and co-amoxiclav in E. coli and is commonly associated with co-carriage of aac(6')-Ib-cr, which narrows aminoglycoside options.

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.

Analysis of Escherichia coli STs and resistance mechanisms in sewage from Islamabad, Pakistan indicates a difference in E. coli carriage types between South Asia and Europe.

Objectives: To discover the Escherichia coli STs and associated resistance mechanisms in the community in Islamabad, Pakistan by analysis of E. coli isolates in sewage. Methods: One hundred and ten E. coli were isolated from sewage across the city of Islamabad without antibiotic bias and confirmed as E. coli by MALDI-TOF MS. Isolates were characterized by fumC/fimH (CH) typing and core-genome MLST. Resistance mechanisms, virulence genes, phylotypes and plasmid incompatibility types were determined in a subset of isolates by in silico analysis. The genomic position of blaCTX-M-15 was determined using S1-PFGE, probing and Nanopore MinION sequencing. Results and conclusions: The most prevalent STs were ST394, ST10 and ST648, accounting for 39% of all isolates collected and were found at many sites across Islamabad. Carbapenemase genes were absent and only a single isolate of ST131 was found. The most prevalent resistance mechanisms were qnrS1 and blaCTX-M-15, with blaCTX-M-15 penetrating many STs and found in 31% of all collected isolates. However, the majority of the successful STs were blaCTX-M-15 negative indicating that resistance is not the main driver of prevalence. Twenty-three percent of blaCTX-M-15 genes were chromosomally encoded and large ISEcp1-mediated insertions included qnrS1 and several plasmid genes. In all chromosomally encoded isolates no plasmid copies of blaCTX-M-15 were found. The most prevalent ST (ST394) contained many enteroaggregative E. coli virulence genes and the fimH30 variant allele previously linked to the success of ST131.

Direct in Gel Genomic Detection of Antibiotic Resistance Genes in S1 Pulsed Field Electrophoresis Gels.

S1 pulsed field gel electrophoresis (PFGE) is a method to separate the bacterial chromosome(s) from plasmid nucleic acids. When combined with ethidium bromide staining and UV visualization this method is excellent at assessing the number of plasmids in individual bacterial strains. It is also good at approximating the true size of each individual plasmid when run against a DNA molecular marker. However, downstream applications such as: the location of individual resistance genes on individual plasmids or the chromosome are hampered by very poor transfer of large DNA molecules from agarose gels to adsorbent nylon or nitrocellulose membranes. Herein, we describe a method to directly probe agarose PFGE gels eliminating the necessity of transfer and generating excellent genomic location results.