Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

A systematic review and meta-analysis of carbapenem resistance and its possible treatment options with focus on clinical Enterobacteriaceae: Thirty years of development in Pakistan.

Background: Carbapenem resistance is epidemic worldwide, these last resort antimicrobials are listed in the WHO 'watch group' with higher resistance potential. During the years 2017-18 Pakistan Antimicrobial Resistance Surveillance System reported an increase in carbapenem resistance. However, a comprehensive information on prevalence and molecular epidemiology of carbapenem resistance in Pakistan is not available. This systematic review and meta-analysis is aimed to report the current carbapenem resistance situation in Pakistan and its treatment options. Methods: In this systematic review and meta-analysis, we investigated the pooled prevalence (PPr) of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae by organizing available data, from Web of Science and PubMed by April 2, 2020, in various groups and subgroups including species, years, provinces, extended spectrum ß-lactamase production, clinical presentation, carbapenemase and metallo-ß-lactamase production, and New Delhi metallo-ß-lactamase (NDM) prevalence. Literature review was updated for the studies publisehd by December 07, 2023. Moreover, we descriptively reviewed the molecular epidemiology of carbapenem resistance in Enterobacteriaceae and non-Enterobacteriaceae in Pakistan. Lastly, we statistically explored different treatment options available for carbapenem resistant infections. We used R package 'metafor' for performing meta-analysis and influence diagnostics and determining treatment options. Results: From two academic databases Web of Science and PubMed we identified 343 studies. Eighty-eight studies were selected for the systematic review and meta-analysis. Seventy-four studies were selected for phenotypic analysis, 36 for genotypic analysis, and 31 for available treatment options. PPr-ID of 12% [0.12 (0.07, 0.16)] was observed for phenotypic carbapenem resistance in Enterobacteriaceae with more prevalence recorded in Klebsiella pneumoniae 24% [0.24 (0.05, 0.44)] followed by 9% [0.09 (-0.03, 0.20)] in Escherichia coli. During the last two decades we observed a striking increase in carbapenem resistance PPr i.e., from 0% [0.00 (-0.02, 0.03)] to 36% [0.36 (0.17, 0.56)]. blaNDM with PPr 15% [0.15 (0.06, 0.23)] in naive isolates was found to be the fundamental genetic determinant for carbapenem resistance in Enterobacteriaceae in Pakistan. Polymyxin B, colistin, tigecycline, and fosfomycin were identified as the suggested treatment options available for multidrug resistant infections not responding to carbapenems. Various studies reported carbapenem resistance from human, animal, and environment sources. Conclusion: In conclusion, we found that NDM-1 producing carbapenem resistant Enterobacteriaceae are increasing in Pakistan. Meta-analysis showed that metallo-ß-lactamases producing E. coli ST405 and K. pneumoniae sequence type11 are the major resistant clones. Number of reported studies in various subgroups and inconsistency in following CLSI guidelines are the potential limitations of this meta-analysis. A National antimicrobial resistance (AMR) surveillance strategy based on One Health is urgently needed to check any future AMR crisis in Pakistan.

Co-existence of NDM-, aminoglycoside- and fluoroquinolone-resistant genes in carbapenem-resistant Escherichia coli clinical isolates from Pakistan.

Background: To determine the prevalence of antimicrobial-resistant genes in carbapenem-resistant Escherichia coli (CRECO). Methods: A total of 290 carbapenem-resistant bacteria were collected from tertiary care hospitals in Lahore (Pakistan). These isolates were confirmed by VITEK 2 and matrix-assisted laser desorption/ionization time of flight. The minimum inhibitory concentration was performed by VITEK 2. Sequence typing, resistant gene identification, DNA hybridization and replicate typing were also performed. Results: 33 out of 290 (11.3%) were CRECO and carried blaNDM; 69, 18 and 12% were NDM-1, NDM-5 and NDM-7, respectively, with 100% resistance to ß-lactams and ß-lactam inhibitors. ST405 and ST468 were mostly identified. NDM-ECO carried approximately 50-450 kb of plasmids and 16 (55%) were associated with IncA/C. Conclusion: NDM-1-producing E. coli are highly prevalent in clinical settings.

Escherichia coli (E. coli) is a type of bacteria found in the gut of humans and animals that causes numerous illnesses such as infection of the blood or urinary tract, diarrhea and vomiting. New Delhi metallo-ß-lactamase (NDM) is a protein produced by E. coli that is capable of breaking down several important antibiotics including penicillins, cephalosporins and carbapenems. E. coli that produce this protein are known as 'resistant', and therefore treatments against these infections are limited. This study looked at how common NDM was found among E. coli taken from hospitalized patients in Lahore, Pakistan, to understand the risks of resistant bacteria in clinical settings and found a high number of a high-risk E. coli.

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.

CTX-M-producing Escherichia coli ST602 carrying a wide resistome in South American wild birds: Another pandemic clone of One Health concern.

Wild birds have emerged as novel reservoirs and potential spreaders of antibiotic-resistant priority pathogens, being proposed as sentinels of anthropogenic activities related to the use of antimicrobial compounds. The aim of this study was to investigate the occurrence and genomic features of extended-spectrum ß-lactamase (ESBL)-producing bacteria in wild birds in South America. In this regard, we have identified two ESBL (CTX-M-55 and CTX-M-65)-positive Escherichia coli (UNB7 and GP188 strains) colonizing Creamy-bellied Thrush (Turdus amaurochalinus) and Variable Hawk (Geranoaetus polyosoma) inhabiting synanthropic and wildlife environments from Brazil and Chile, respectively. Whole-genome sequence (WGS) analysis revealed that E. coli UNB7 and GP188 belonged to the globally disseminated clone ST602, carrying a wide resistome against antibiotics (ß-lactams), heavy metals (arsenic, copper, mercury), disinfectants (quaternary ammonium compounds), and pesticides (glyphosate). Additionally, E. coli UNB7 and GP188 strains harbored virulence genes encoding hemolysin E, type II and III secretion systems, increased serum survival, adhesins and siderophores. SNP-based phylogenomic analysis, using an international genome database, revealed genomic relatedness (19-363 SNP differences) of GP188 with livestock and poultry strains, and genomic relatedness (61-318 differences) of UNB7 with environmental, human and livestock strains (Table S1), whereas phylogeographical analysis confirmed successful expansion of ST602 as a global clone of One Health concern. In summary, our results support that ESBL-producing E. coli ST602 harboring a wide resistome and virulome have begun colonizing wild birds in South America, highlighting a potential new reservoir of critical priority pathogens.

The Diversity, Resistance Profiles and Plasmid Content of Klebsiella spp. Recovered from Dairy Farms Located around Three Cities in Pakistan.

The rise of antimicrobial resistance (AMR) in bacterial pathogens such as Klebsiella pneumoniae (Kp) is a pressing public health and economic concern. The 'One-Health' framework recognizes that effective management of AMR requires surveillance in agricultural as well as clinical settings, particularly in low-resource regions such as Pakistan. Here, we use whole-genome sequencing to characterise 49 isolates of Klebisella spp. (including 43 Kp) and 2 presumptive Providencia rettgeri isolates recovered from dairy farms located near 3 cities in Pakistan-Quetta (n = 29), Faisalabad (n = 19), and Sargodha (n = 3). The 43 Kp isolates corresponded to 38 sequence types (STs), and 35 of these STs were only observed once. This high diversity indicates frequent admixture and limited clonal spread on local scales. Of the 49 Klebsiella spp. isolates, 41 (84%) did not contain any clinically relevant antimicrobial resistance genes (ARGs), and we did not detect any ARGs predicted to encode resistance to carbapenems or colistin. However, four Kp lineages contained multiple ARGs: ST11 (n = 2), ST1391-1LV (n = 1), ST995 (n = 1) and ST985 (n = 1). STs 11, 1391-1LV and 995 shared a core set of five ARGs, including blaCTX-M-15, harboured on different AMR plasmids. ST985 carried a different set of 16 resistance genes, including blaCTX-M-55. The two presumptive P. rettgeri isolates also contained multiple ARGs. Finally, the four most common plasmids which did not harbour ARGs in our dataset were non-randomly distributed between regions, suggesting that local expansion of the plasmids occurs independently of the host bacterial lineage. Evidence regarding how dairy farms contribute to the emergence and spread of AMR in Pakistan is valuable for public authorities and organizations responsible for health, agriculture and the environment, as well as for industrial development.

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.

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.

The Prevalence of Plasmid-Mediated Colistin Resistance Gene mcr-1 and Different Transferability and Fitness of mcr-1-Bearing IncX4 Plasmids in Escherichia coli from Pigeons.

The prevalence of colistin-resistant bacteria limited the usage of colistin in the treatment of clinical multidrug-resistant Gram-negative bacterial infections. Here, we aimed to investigate the prevalence and molecular characterization of mcr-1-carrying isolates from pigeons close to humans following the ban on the use of colistin as an animal feed additive in China. Methods, including PCR, antimicrobial susceptibility testing, conjugation experiments, plasmid replicon typing, genome sequencing, bioinformatics analysis, measurement of growth curves, competition experiments, and plasmid stability assays were used to identify and characterize mcr-1-positive isolates. In total, 45 mcr-1-positive E. coli isolates were acquired from 100 fecal samples, and MICs of colistin ranged from 4 to 8 mg/L. The prevalence of mcr-1-positive E. coli isolates from pigeons was mainly mediated by IncX4 plasmids (39/45), including transferable mcr-1-bearing IncX4 plasmids with fitness advantage in 21 isolates, and nontransferable mcr-1-bearing IncX4 plasmids with fitness disadvantage in 18 isolates. There is a similar structure among the 6 mcr-1-bearing nontransferable IncX4 plasmids and 10 mcr-1-bearing transferable IncX4 plasmids in 16 E. coli isolates that have been sequenced. Plasmid transferability evaluation indicated that the same IncX4 plasmid has different transferability in different E. coli isolates. In conclusion, this study demonstrates that pigeons could act as potential reservoirs for the spread of mcr-1-positive E. coli in China. Transferability of IncX4 plasmids may be influenced by host chromosome in the same bacterial species. Additional research on the factors influencing the transferability of IncX4 plasmids in different bacterial hosts is required to help combat antimicrobial resistance. IMPORTANCE The emergence of plasmid-mediated colistin resistance gene mcr-1 incurs great concerns. Since the close proximity of pigeons with humans, it is significant to understand the prevalence and molecular characterization of mcr-1-positive isolates in pigeons, to provide a rationale for controlling its spread. Here, we found that the prevalence of mcr-1-positive E. coli from pigeons was mainly mediated by IncX4 plasmids. However, different transferability and fitness of mcr-1-bearing IncX4 plasmids in E. coli were observed, which demonstrated that transferability of IncX4 plasmids could be affected not only by genes on plasmids, but also by chromosomal factors in the same bacterial species. Our finding provided a new insight on studying the factors influencing the transferability of plasmids.

Association between antimicrobial consumption and resistance rate of Escherichia coli in hospital settings.

AIMS: This study aims to quantify antimicrobial consumption (AMC) and antimicrobial resistance (AMR) in tertiary care hospitals in Pakistan. METHODS AND RESULTS: This observational study was conducted at two tertiary care hospitals of Pakistan over a 1-year period. The AMC and resistance patterns of Escherichia coli isolates collected from hospitals were observed and analysed using the pearson correlation coefficient. AMC in hospitals varied between 0.00186 and 0.72 (Mean = 0.12 ± 0.191) Defined Daily Dose (DDDs)/1000 patient-days. Fluoroquinolones were the most consumed antimicrobial followed by penicillins, cephalosporins, carbapenems, and aminoglycosides. Antimicrobial-resistant rates in hospitals varied between 92.2% and 34.6%. The highest resistance rate was observed for moxifloxacin, followed by ampicillin, cefotaxime, amoxicillin, ceftriaxone, ciprofloxacin, amoxiclav, and amikacin. Statistically significant association was found between AMC and resistance rate for ampicillin (r = 0.78, P = 0.032), cefotaxime (r = 0.87, P = 0.012), ceftriaxone (r = 0.67, P = 0.042), and ciprofloxacin (r = 0.63, P = 0.031). Additionally, there was a significant association between fluoroquinolone consumption and the resistance rate of third generation cephalosporins (r = 0.61, P = 0.032), and significance was also found when all antimicrobials were combined into 1 analysis (r = 0.721, P = 0.032). CONCLUSION: This data documented a significant association between AMC and resistant rates for multiple antimicrobial agents.

Veterinary consumption of highest priority critically important antimicrobials and various growth promoters based on import data in Pakistan.

BACKGROUND: Antimicrobial resistance (AMR) is a global public health emergency driven by the indiscriminate use of antimicrobial agents in humans and animals. Antimicrobial consumption surveillance guides its containment efforts. In this study, we estimated, for the first time, veterinary consumption of Critically Important Antimicrobials with Highest Priority (CIA-HtP) for Pakistan. METHODS: The study used an export/import database which provided imports data collected from the Pakistan Customs Authority. We investigated imports of 7 CIA-HtP and various poultry feed additives/growth promoters (FAs/GPs) identified from a survey of 10 poultry and dairy farms in Punjab province in Pakistan and a previously published study, over a three-year period of 2017-2019. Antimicrobial consumption was estimated in mg/kg of country's animal biomass. FINDINGS: Imports, in tonnes, for these 7 CIA-HtP were for the years 2017-19: tylosin 240.84, enrofloxacin 235.14, colistin 219.73, tilmicosin 97.32, spiramycin 5.79, norfloxacin 5.55, ceftiofur 1.02 for a total 805.39 tonnes. The corresponding antimicrobial consumption was 10.05 mg/kg of animal biomass. The poultry FAs/GPs contained: zinc bacitracin, enramycin, bacitracin methylene disalicylate, tylosin, tiamulin, colistin, lincomycin, streptomycin, flavophospholipol, tilmicosin, and penicillin with a total antimicrobial chemical compound (ACC) import volume of 577.18 tonnes for the years 2017-2019; and an estimated consumption of 96.53 mg/kg of poultry biomass. INTERPRETATION: These antimicrobials were a mix of macrolides, quinolones, polymyxins and cephalosporins, among which are some also on the Watch or Reserve list by the WHO, indicating the need for stewardship and to conserve essential antimicrobials to contain AMR. The finding that a yearly average of 192.39 tonnes of the ACC imported were FAs/GPs further highlight the need for stronger regulation and enforcement.

Emergence and Characterization of Tigecycline Resistance Gene tet(X4) in ST609 Escherichia coli Isolates from Wastewater in Turkey.

Emergence of pathogens harboring tigecycline resistance genes incurs great concerns. Wastewater is recognized as the important reservoir of antimicrobial resistance genes. Here we characterized the phenotypes and genotypes of bacteria carrying tet(X4) from wastewater in Turkey for the first time. Four tet(X4)-positive Escherichia coli isolates were identified and characterized by PCR, Sanger sequencing, antimicrobial susceptibility testing, conjugation assays, Illumina sequencing, nanopore sequencing and bioinformatic analysis. Four tet(X4)-harboring isolates were multidrug-resistant (MDR) bacteria and the tet(X4) gene was nontransferable in four isolates. Genetic analysis revealed that tet(X4) genes in four isolates were located on plasmids co-harboring two replicons IncFIA(HI1) and IncFIB(K). However, none of the four plasmids carried genes associated with horizontal transfer of plasmids. The coexistence of blaSHV-12-bearing IncX3-type plasmid and tet(X4)-harboring plasmid was also found in one isolate. These findings indicate that continuous surveillance of the tet(X4)-bearing isolates in different environments worldwide should be strengthened. IMPORTANCE The emergence of tigecycline resistance genes in humans and animals in China seriously threatens the clinical utility of tigecycline, but the molecular epidemiology of tigecycline-resistant bacteria in other countries remained largely unknown. Therefore, it is necessary to learn the prevalence and molecular characteristics of bacteria carrying tigecycline resistance genes, particularly the mobilizable tet(X4), in other countries. In the study, we first described the presence and molecular characteristics of the tet(X4)-positive E. coli isolates from wastewater in Turkey. Four tet(X4)-bearing isolates belonged to ST609, an E. coli clone commonly found from humans, animals and the environment. These findings highlight the importance of monitoring the tet(X4) gene in different settings globally.

Pathological effects of graded doses of aflatoxin B1 on the development of the testes in juvenile white leghorn males.

Current experiment was planned to investigate the deleterious effects of the graded doses of aflatoxin B1 (AFB1) on white leghorn male birds. For this purpose, one-hundred birds of 8 weeks of age were divided into 4 equal groups and reared on feed contaminated with different doses of AFB1 for 10 weeks. Group A was kept as a control group and was fed with normal toxin-free diet; groups B, C, and D were offered feed containing 100 ppb, 200 ppb, and 400 ppb of AFB1, respectively. The birds were euthanized at the 4th and 10th week of the experiment. Clinical signs, behavioral changes, absolute and relative organ weight of the testes, and sperm motility were measured. Cellular immune response was observed through carbon clearance assay (CCA), P-HAP, and antibody response against sheep red blood cells (SRBC). Results showed a dose-dependent decline in the immune response of birds with the increase in the level of AFB1 in the feed. A significant decrease in the serum levels of testosterone, prolactin, and LH were observed at the end of the study. Grossly, testicular size and volume were reduced in ABF1 fed birds, while histological examination showed moderate to severe necrosis of testicular parenchyma, with partial to complete arrest of spermatogenesis. Very few spermatozoa were found in group C, while they were almost absent in group D which was offered a diet containing 400 ppb AFB1. The motility of sperms was reduced in all treated groups except control. The abovementioned results showed that AFB1 had severe toxic effects on the reproductive and immunological parameters of WLH male birds in a dose-dependent manner.

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 ß-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 blaNDM-1 -producers was assayed by conjugation experiments. All ESBL strains carried the blaCTX-M-15 gene. Of these blaCTX-M-15 producing E. coli, four also carried blaNDM-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 blaNDM-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.

Genomically diverse carbapenem resistant Enterobacteriaceae from wild birds provide insight into global patterns of spatiotemporal dissemination.

Carbapenem resistant Enterobacteriaceae (CRE) are a threat to public health globally, yet the role of the environment in the epidemiology of CRE remains elusive. Given that wild birds can acquire CRE, likely from foraging in anthropogenically impacted areas, and may aid in the maintenance and dissemination of CRE in the environment, a spatiotemporal comparison of isolates from different regions and timepoints may be useful for elucidating epidemiological information. Thus, we characterized the genomic diversity of CRE from fecal samples opportunistically collected from gulls (Larus spp.) inhabiting Alaska (USA), Chile, Spain, Turkey, and Ukraine and from black kites (Milvus migrans) sampled in Pakistan and assessed evidence for spatiotemporal patterns of dissemination. Within and among sampling locations, a high diversity of carbapenemases was found, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-beta-lactamase (NDM), oxacillinase (OXA), and Verona integron Metallo beta-lactamase (VIM). Although the majority of genomic comparisons among samples did not provide evidence for spatial dissemination, we did find strong evidence for dissemination among Alaska, Spain, and Turkey. We also found strong evidence for temporal dissemination among samples collected in Alaska and Pakistan, though the majority of CRE clones were transitory and were not repeatedly detected among locations where samples were collected longitudinally. Carbapenemase-producing hypervirulent K. pneumoniae was isolated from gulls in Spain and Ukraine and some isolates harbored antimicrobial resistance genes conferring resistance to up to 10 different antibiotic classes, including colistin. Our results are consistent with local acquisition of CRE by wild birds with spatial dissemination influenced by intermediary transmission routes, likely involving humans. Furthermore, our results support the premise that anthropogenically-associated wild birds may be good sentinels for understanding the burden of clinically-relevant antimicrobial resistance in the local human population.

Widespread prevalence and molecular epidemiology of tet(X4) and mcr-1 harboring Escherichia coli isolated from chickens in Pakistan.

The emergence and spread of plasmid-mediated tigecycline resistance gene tet(X4) and colistin resistance gene mcr-1 in Escherichia coli (E. coli) pose a potential threat to public health, due to the importance of colistin and tigecycline for treating serious clinical infections. However, the characterization of bacteria coharboring both genes was few reported. Here, we described the molecular epidemiology of tet(X4) and mcr-1 harboring E. coli strains of chicken origin in Pakistan, with methods including PCR, antimicrobial susceptibility testing, DNA transfer assays, plasmid replicon typing, whole-genome sequencing and bioinformatics analysis. The tet(X4) gene was identified in 36 isolates exhibiting high levels of tigecycline resistance (MICs, 16-128 mg/L). Worryingly, 24 of the 36 tet(X4)-bearing isolates were confirmed as colistin resistance, positive for plasmid-borne mcr-1. We observed the prevalence of tet(X4)-bearing IncFII plasmid with mcr-1-bearing IncI2 plasmid in 12 E. coli isolates, with a high co-transfer frequency except for one strain PK8233, in which tet(X4)- and mcr-1-bearing plasmids were non-transferable. Coexistence of tet(X4)-bearing IncFII plasmid with mcr-1-carrying multidrug-resistant (MDR) IncHI2 plasmid was also identified in 10 E. coli isolates, and a relatively low co-transfer frequency was obtained except PK8575, in which mcr-1 was non-transferable. The transferability of pPK8275-tetX in PK8275 and pPK8233-tetX in PK8233, that could transfer from E. coli J53 to C600 by conjugation, was interfered by certain factors in PK8275 and PK8233. This may provide new insights to prevent and control the spread of antibiotic resistance genes. Two strains were reported to co-carry tet(X4)-positive IncQ1 plasmid and mcr-1-positive IncI2 plasmid. Convergence of tet(X4) and mcr-1 genes in E. coli by conjugative or mobilizable plasmids may lead to potentially widespread transmission of such resistance genes, which may incur antibiotic-resistance crisis globally.

Measuring Antimicrobial Use Needs Global Harmonization.

Global health and global economies are predicted to be severely affected by antimicrobial resistance (AMR). The three organizations World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) are working in their domains to prevent any future AMR crisis. Antimicrobial use (AMU), especially in food animals, is contributing to the development and dissemination of AMR bacteria and genes. AMU monitoring is a strategic objective of the global and national action plans on AMR. However, the AMU reporting metrics at different levels are not harmonized yet, posing difficulties in comparisons among AMU data from different sources. A tripartite WHO/OIE/FAO collaboration is urgently required to develop and implement a globally accepted AMU metric system to ensure reliable comparisons among various data sets.