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

Carbapenem resistance among Enterobacterales has become a global health concern. Clinical Escherichia coli isolates producing the metallo β-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. IMPORTANCE We 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

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

The role of IL-2 in the pathogenesis of multiple myeloma “a study in Iraqi patients”

IL-2 plays a pivotal role in the induction of host immune responses against tumor growth and it has anti-tumor activity. Therefore the present study aimed at shedding light on a group of Iraqi patients with multiple myeloma by evaluating the level of this cytokine in their sera . The age and gender were also taken into consedration. and investigate if there was any association between IL-2 and the level of β2-m. A group of 49 patients and 22 healthy volunteers were included in the study. The quantitative determination of IL-2 serum level was performed by ELISA. The patients were divided into two groups: the first group included (17) patients who were recently diagnosed and not received any treatment at the time of collecting samples, while the second group included (32) patients who received treatment

High Prevalence of CTX-M-15-Type ESBL-Producing E. coli from Migratory Avian Species in Pakistan

The increased presence of clinically relevant multidrug resistant bacteria in natural environments is an emerging challenge for global health care. Little is known regarding the occurrence of extended-spectrum beta-lactamase producing Escherichia coli (ESBL-E. coli) from environmental sentinels in Pakistan. The goal of the current study was to gain insights into the prevalence and phylogenetic relationships of ESBL-E. coli recovered from wild birds in Pakistan during winter migration. After initial screening of fecal samples on selective chromogenic agar, ESBL-E.coli were analyzed phenotypically using the Vitek-2 automated system. Genotypic characterization was performed using whole genome sequencing (WGS) followed by an in-depth in silico analysis. Of 150 birds screened, 26 (17.3%) were fecal carriers of ESBL-E. coli. Of these, 88.4% isolates exhibited multidrug resistance (MDR) phenotypes. Resistance to cefotaxime, ceftazidime, ampicillin, doxycycline, tetracycline and sulfamethoxazole/trimethoprim (CTX-CAZ-AM-DC-TE-SXT) represented the most common pattern of MDR (76.9%). WGS data analysis found blaCTX-M-15 as the predominant ESBL genotype (92.3%). Other genes encoding resistance to sulfonamides (sul1/sul2/sul3), aminoglycosides (strA, strB, aadA1, aadA2, aadA5, aac(3)-IId-like, aac(3)-IVa-like and aph(4)-Ia), trimethoprim (dfrA14 or dfrA17), tetracyclines [tet(A)/tet(B)], and fluoroquinolones (qnrS1) were detected commonly, often encoded on IncF-type plasmids (76.9%). ESBL-E. coli were assigned to 17 different sequence types (STs) of which ST10 and ST7097 (4 isolates each) were the most abundant followed by ST4720, ST93, and ST1139 (2 isolates each). Core-genome phylogeny of the isolates found low numbers (0–29) of single nucleotide polymorphisms (SNPs) in isolates belonged to ST7097 originated from two different locations (Chashma barrage and Rasul barrage). Similar trends were found among isolates belong to ST1139. In addition, WGS-based plasmid typing and S1-digestion found plasmids of the same pMLST type (IncF[F-:A-:B53]) and similar sizes in different bacterial and avian hosts suggesting horizontal gene transfer as another possibility for the spread of ESBL-E. coli in avian wildlife in Pakistan

Case Study: Using a Shared International Database to Document Veterinary Consumption of Antibiotics in Pakistan

In this paper, we present a case study of Pakistan documenting the use of antimicrobial drugs in poultry flocks in the VetCAb-ID database. Unlike other databases, this system allows international users to upload their data directly. Based on expert interviews and a review of the latest publications on the topic, we provide an alternative approach to harmonizing data collection among countries. This paper will provide impetus to formulate joint requirement documentation for an AMU database on a global level that international users can adapt for their own purposes and projects

Genomic characterization of high-risk Escherichia coli and Enterobacter hormaechei clones recovered from a single tertiary-care hospital in Pakistan

Aims: Spread of carbapenem-resistant Enterobacterales have become a global problem. We characterized extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales from urinary tract infections cases from Allied Hospital Faisalabad, Pakistan. Methods and Results: Eleven (22%, 11/50) ESBL-producing Enterobacterales (Escherichia coli; n = 10 and Enterobacter hormaechei; n = 1) were recovered and processed through VITEK-2, PCR, rep-PCR followed by whole-genome sequencing (WGS) of ESBL-producing Ent. hormaechei and carbapenem-resistant E. coli isolates. Plasmid transferability of bla(NDM-1)-producers was assayed by conjugation experiments. All ESBL strains carried the bla(CTX-M-15) gene. Of these bla(CTX-M-15) producing E. coli, four also carried bla(NDM-1) located on transferable plasmids. All E. coli strains belonged to ST448 and displayed similar genetic features including genes for antimicrobial resistance, heavy metal, biocides and virulence. Genomic features of a multidrug-resistant (MDR) Ent. hormaechei were also reported for the first time in Pakistan. Conclusion: Our findings indicate that bla(NDM-1) producing E. coli ST448 is a multidrug, heavy metals and biocides-resistant strain. Therefore, the screening of these isolates may be effective in limiting the MDR bacteria spread in hospitalized patients and within the community. Significance and Impact of this Study: Spread of multi-drug-resistant ESBL-producing bacteria in the clinical settings of Pakistan is a serious challenge and further limiting treatment options in the country. WGS could be used as a tool in the nationwide antibiotic surveillance programme to explore insights of spread and outbreak

Emergence of extended spectrum beta-lactamases-producing strains belonging to cefotaxime-M-1 class from intensive care units patients and environmental surfaces in Pakistan

Aim: The emergence of multidrug-resistant (MDR) bacteria is the most dangerous threat for the treatment of infectious diseases. The aim of this study was to detect and characterize extended spectrum beta-lactamases (ESBLs) and carbapenemase-producing Klebsiella pneumoniae and Escherichia coli among patients and environment of intensive care units (ICUs) of three tertiary care hospitals in Pakistan. Materials and Methods: A total of 82 samples from ICU’s patients and inanimate environment (injection trays, wash basins, door handles, hand swabs of professionals, and ICU fridges) were screened for ESBL by culturing on CHROMagar-ESBL. ESBL and carbapenemases production were confirmed by double disc synergy test and modified Hodge’s test, respectively. Polymerase chain reaction was used to detect ESBL encoding genes bla cefotaxime (CTX-M), blaCTX-M-1, blaCTX-M-2, blaCTX-M-9, blaTEM, blaSHV and carbapenemase genes blaKPC, bla New Delhi metallo-beta-lactamase-1, blaOXA-48 and blaVIM. Results: Overall, ESBL production was found high 30/82 (36.5%) among isolates of which 15.8% K. pneumoniae and 20.7% E. coli were identified. All the K. pneumoniae and majority of E. coli isolates were MDR, i.e., resistance to three or more antimicrobial categories. Molecular characterization showed the blaCTX-M-1 as the predominant genotype found in 17/21 (80%) of the isolates. None of the strains was found positive for carbapenemase-encoding genes. Conclusion: In conclusion, this study demonstrates the emergence of MDR ESBL producing strains among ICU patients and hospital environment, posing a serious threat for the control of nosocomial infections