Multidrug-resistant Enterobacteriaceae Research Articles

Sub-MIC Antibiotics Modulate Productions of Outer Membrane Vesicles in Tigecycline-Resistant Escherichia coli.

Antimicrobial resistance (AMR) has been recognized as one of the most important crises affecting global human health in the 21st century. Tigecycline is one of the last resort antibiotics for treating severe infections caused by multi-drug resistant Enterobacteriaceae. However, the mobile resistance gene tet(X4), which could mediate high-level tigecycline resistance, was discovered in 2019. The outer membrane vesicle (OMV) has been recognized as a new route for horizontal gene transfer; antimicrobial resistant bacteria also have the ability to secret OMVs, while little is known about the impact of antibiotics on the secretion and characteristics of OMVs from tigecycline resistant bacteria till now. This study aimed to investigate the effects of antibiotics on the production and traits of a tigecycline resistant Escherichia coli strain of 47EC. The results showed that sub-inhibitory (1/2 MIC or 1/4 MIC) concentrations of gentamicin, meropenem, ceftazidime, chloramphenicol, tigecycline, ciprofloxacin, polymycin, rifaximin and mitomycin C could significantly increase the secretion of OMVs (0.713 ± 0.05~6.333 ± 0.15 mg/mL) from E. coli 47EC compared to the respective untreated control (0.709 ± 0.03 mg/mL). In addition, the particle sizes of OMVs were generally larger, and the zeta potential were lower in the antibiotics-treated groups than those of the antibiotic-free group. The copy numbers of the tigecycline resistance gene of tet(X4) in the OMVs of most antimicrobial-treated groups were higher than that of the control group. Moreover, transcriptome analysis on ciprofloxacin-treated E. coli 47EC indicated that the SOS response and prophage activation might participate in the ciprofloxacin-induced OMV formation. In conclusion, the clinical application of antibiotics in treating bacterial infections, especially multi-drug resistant bacteria, might lead to the increased secretion of bacterial OMVs and the enrichment of antimicrobial-resistant genes in the OMVs.

Transposition mechanism of ISApl1-the determinant of colistin resistance dissemination.

Multidrug-resistant Enterobacteriaceae, a prominent family of gram-negative pathogenic bacteria, causes a wide range of severe diseases. Strains carrying the mobile colistin resistance (mcr-1) gene show resistance to polymyxin, the last line of defense against multidrug-resistant gram-negative bacteria. However, the transmission of mcr-1 is not well understood. In this study, genomes of mcr-1-positive strains were obtained from the NCBI database, revealing their widespread distribution in China. We also showed that ISApl1, a crucial factor in mcr-1 transmission, is capable of self-transposition. Moreover, the self-cyclization of ISApl1 is mediated by its own encoded transposase. The electrophoretic mobility shift assay experiment validated that the transposase can bind to the inverted repeats (IRs) on both ends, facilitating the cyclization of ISApl1. Through knockout or shortening of IRs at both ends of ISApl1, we demonstrated that the cyclization of ISApl1 is dependent on the sequences of the IRs at both ends. Simultaneously, altering the ATCG content of the bases at both ends of ISApl1 can impact the excision rate by modifying the binding ability between IRs and ISAPL1. Finally, we showed that heat-unstable nucleoid protein (HU) can inhibit ISApl1 transposition by binding to the IRs and preventing ISAPL1 binding and expression. In conclusion, the regulation of ISApl1-self-circling is predominantly controlled by the inverted repeat (IR) sequence and the HU protein. This molecular mechanism deepens our comprehension of mcr-1 dissemination.

Antibacterial and antibiofilm activities of Thymus leptobotrys and Lavandula mairei essential oils against extended spectrum β-lactamase producing Enterobacteriaceae

The aim of the study was to investigate the antibacterial and antibiofilm activities of Lavandula mairei and Thymus leptobotrys essential oils against Extended spectrum β-lactamase producing Enterobacteriaceae, responsible of nosocomial infections. PCR and sequencing methods detected three different resistance genes (blaSHV-42, blaSHV-12 et blaCTXM-15) in the studied strains. Gas chromatography-mass spectrometry analysis highlighted the predominance of carvacrol in both Lavandula mairei and Thymus leptobotrys essential oils. Their antibacterial activity was conducted by disk diffusion and microdilution methods. Great sensitivity of the multidrug resistant strains was noted for the two essential oils with inhibition zones’ diameters ranging from 13.3±2.3 to 32.3±1.4 mm (Thymus leptobotrys) and from 22 to 34.66±0.57 mm (Lavandula mairei), and low minimal and bactericidal concentrations varying from 1.56 to 6.25 mg/mL (CMI) and from 1.56 to 12.5 mg/mL (CMB) for both essential oils. Four strains out of six, considered as the strongest biofilm producers by congo red agar and crystal violet methods, were chosen to evaluate biofilm inhibition and eradication effects of Lavandula mairei and Thymus leptobotrys essential oils. In most cases, these latter inhibited considerably the biofilm formation of the targeted strains with proportions greater than 80%. The eradication effect was lower, with rates not higher than 50%. Thus, these essential oils could be used as an effective alternative approach to prevent biofilm formation of multidrug resistant Enterobacteriaceae. To our knowledge, no previous works have reported the antibiofilm activity of Lavandula mairei and Thymus leptobotrys essential oils against these multidrug resistant bacteria.

Multidrug-Resistant ESBL-Producing Enterobacteriaceae Associated with Clinical Samples in a Tertiary Care Hospital, Sirajganj

Extended-spectrum β-lactamase (ESBL) microorganisms have been shown to increase multidrug resistance worldwide, which is a great concern. The prevalence of ESBL-producing clinical pathogens and their antimicrobial resistance patterns were identified in 66 isolates from patients in Khwaja Yunus Ali Hospital with several clinical infections cultured on blood agar and MacConkey agar media. The most prevalent bacteria were Escherichia coli (80.3%), which were identified by the BD-Phonex automated identifier machine, followed by Pseudomonas spp. (6%), Klebsiella spp. (12.1%), and others (4.4%). This research was conducted from January 2023 to June 2023. Generally, a large number of antibiotic resistance patterns and ESBL-producing common bacterial isolates were found in this study, where most of the resistant percentage was found in third-generation cephalosporin antibiotics, which increases the public health problem. In this study, the most prevalent bacteria E. coli isolates were mostly resistant to penicillin (100%), ceftazidime, cefotaxime, and cefuroxime (98.4%). Besides, 95.4% resistance was shown against ceftriaxone. The double-disc synergy test was done to determine the presence of ESBL-producing bacterial strains. The most widely ESBL-positive isolate was Escherichia coli (83%). Among the 66 sample strains produced, the ESBL maximum (53.03%) belonged to female patients, while 46.97% belonged to male patients. This study focuses on the prevalence and patterns of clinical pathogens and the antimicrobial susceptibility profiles of ESBL-producing bacterial infections in a tertiary-level health service center in Bangladesh. Generally, a large number of antibiotic resistance patterns and ESBL-producing common bacterial isolates were found in this study, which increases the public health risk. Therefore, to save human life, we ought to be taking appropriate action against the threat.

Punicalagin, a pomegranate polyphenol sensitizes the activity of antibiotics against three MDR pathogens of the Enterobacteriaceae

BackgroundMultidrug resistance (MDR) in the family Enterobacteriaceae is a perniciously increasing threat to global health security. The discovery of new antimicrobials having the reversing drug resistance potential may contribute to augment and revive the antibiotic arsenal in hand. This study aimed to explore the anti-Enterobacteriaceae capability of bioactive polyphenols from Punica granatum (P. granatum) and their co-action with antibiotics against clinical isolates of Enterobacteriaceae predominantly prevalent in South Asian countries.MethodsThe Kandhari P. granatum (Pakistani origin) extracts were tested for anti-Enterobacteriaceae activity by agar well diffusion assay against MDR Salmonella enterica serovar Typhi, serovar Typhimurium and Escherichia coli. Predominant compounds of active extract were determined by mass spectrometry and screened for bioactivity by agar well diffusion and minimum inhibitory concentration (MIC) assay. The active punicalagin was further evaluated at sub-inhibitory concentrations (SICs) for coactivity with nine conventional antimicrobials using a disc diffusion assay followed by time-kill experiments that proceeded with SICs of punicalagin and antimicrobials.ResultsAmong all P. granatum crude extracts, pomegranate peel methanol extract showed the largest inhibition zones of 25, 22 and 19 mm, and the MICs as 3.9, 7.8 and 7.8 mg/mL for S. typhi, S. typhimurium and E. coli, respectively. Punicalagin and ellagic acid were determined as predominant compounds by mass spectrometry. In plate assay, punicalagin (10 mg/mL) was active with hazy inhibition zones of 17, 14, and 13 mm against S. typhi, S. typhimurium and E. coli, respectively. However, in broth dilution assay punicalagin showed no MIC up to 10 mg/mL. The SICs 30 μg, 100 μg, and 500 μg of punicalagin combined with antimicrobials i.e., aminoglycoside, β-lactam, and fluoroquinolone act in synergy against MDR strains with % increase in inhibition zone values varying from 3.4 ± 2.7% to 73.8 ± 8.4%. In time-kill curves, a significant decrease in cell density was observed with the SICs of antimicrobials/punicalagin (0.03–60 μg/mL/30, 100, 500 μg/mL of punicalagin) combinations.ConclusionsThe P. granatum peel methanol extract exhibited antimicrobial activity against MDR Enterobacteriaceae pathogens. Punicalagin, the bacteriostatic flavonoid act as a concentration-dependent sensitizing agent for antimicrobials against Enterobacteriaceae. Our findings for the therapeutic punicalagin-antimicrobial combination prompt further evaluation of punicalagin as a potent activator for drugs, which otherwise remain less or inactive against MDR strains.

Molecular characterization of extended-spectrum beta-lactamases and carbapenemases producing Enterobacteriaceae isolated from North Eastern region of India

Objectives: This study is aimed to investigate the prevalence of genes encoding extended-spectrum β-lactamases (ESBLs) and carbapenemases production among Enterobacteriaceae isolated from North East India. Materials and Methods: A total of 210 non-duplicate multi-drug resistant Enterobacteriaceae (MDRE) strains were included in this investigation. The isolates were resistant to third-generation cephalosporins, aminoglycosides, and fluoroquinolones. First, the strains were subjected to phenotypic assays to determine ESBLs and carbapenemases production; then, multiplex polymerase chain reaction (mPCR) assays were done to detect ESBLs and carbapenemases genes. In addition, efflux pump activity was determined by phenylalanine-arginine b-naphthylamide assay. Statistical Analysis: The frequency of ESBLs and carbapenemase genes among MDRE strains was shown as percentages. The data analysis was done using Microsoft Excel computer software. Results: Among 210 MDRE clinical isolates, ESBLs production was observed in 72.86% (153) isolates. During mPCR assay, gene encoding ESBLs were detected in 55.24% (116) MDRE strains beta-lactamase Temoniera (blaTEM) (26.67%, 56), beta-lactamase Cefotaxime-Munich (blaCTX-M) (19.52%, 41), and beta-lactamase sulfhydryl reagent variable (blaSHV) (9.05%, 19)]. In addition, 55 (26.2%) and 53 (25.26%) strains were found to be meropenem and imipenem resistant, respectively. Carbapenemase nordmann-poirel (Carba-NP) test for carbapenemases activity was found to be positive in 18.58% (39) MDRE strains. The genes encoding carbapenemases production was observed in 18.58% (39) MDRE [beta-lactamase New Delhi metallo-β-lactamases-1(blaNDM-1) (8.10%, 17), beta-lactamase oxacillinase-48 (blaOXA-48) (2.86%, 6), beta-lactamase Verona imipenemase (blaVIM) (1.43%, 3), and blaOXA-48 and blaVIM (6.19%, 13)]. Efflux pump activity was observed in 5 (2.3%) of Carbapenem-resistant Enterobacteriaceae isolates. Conclusions: For the first time in this region, we have detected the presence of blaOXA-48 and blaVIM in a single MDRE isolate as high as 6.1%. Therefore, clinicians need to detect the ESBLs and carbapenemases producing Enterobacteriaceae on priority in hospital settings for therapeutic options as well as stringent infection control strategies to be adopted as precautions.

Carvacrol-loaded invasomes efficacy against multidrug resistant isolates of Enterobacteriaceae and housefly

The current study aimed to evaluate the antimicrobial activity of carvacrol-loaded invasomes (CLI) against multidrug-resistant (MDR) Enterobacteriaceae and its mechanical vector, the housefly. CLI were prepared and characterized in the laboratory. Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serovar Enteritidis, Salmonella enterica subsp. enterica serovar Typhimurium, and Klebsiella oxytoca were among the MDR enterobacteriaceae stains investigated. These strains were first isolated and identified from naturally infected chickens. The antibacterial activity of CLI against the MDR isolates was evaluated using the diffusion method. In addition, the insecticidal activity of CLI against housefly larvae and pupae was tested. The MDR index of all evaluated isolates was greater than 20%, indicating that they were all multidrug-resistant. CLI decreased the growth of all isolates except S. Typhimurium and P. aeruginosa at a dose of 0.0125%; however, pure carvacrol inhibited the growth of only Klebsiella oxytoca. Furthermore, both CLI and pure carvacrol inhibited Klebsiella oxytoca growth at different concentrations. CLI inhibited E. coli and S. enteritidis at lower concentrations than pure carvacrol, even at a doubled concentration. Carvacrol and CLI caused significant larval mortality even at low concentrations, with LC50 reached at concentartions of 2.54 and 2.19 µl/ml, respectively. Furthermore, at a low concentration of 3.125 µl/ml, both elicited a high percentage inhibition rate (PIR) in pupae. In conclusion, CLI demonstrated substantial antibacterial action, particularly against MDR isolates, as well as pesticide activity against houseflies.  

Assessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal.

Clinicians face a global challenge treating infections caused by Enterobacteriaceae because of the high rate of antibiotic resistance. This cross-sectional study from the Nepal Armed Police Force Hospital, Kathmandu, Nepal, characterized resistance patterns in Enterobacteriaceae across different antimicrobial classes and assessed incidences of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections. Enterobacteriaceae from clinical samples were isolated on blood and MacConkey agar, except for urine samples on cysteine lactose electrolyte-deficient agar. To determine antimicrobial susceptibility patterns, including MDR and XDR, the Kirby-Bauer disc diffusion method was used. Statistics were performed using SPSS, v. 17.0. Members of the family were identified in 14.5% (95% CI: 16.2-12.8%) of the total samples (N = 1,617), primarily in urine (54.7%, 128/234), blood (19.7%, 46/234), and sputum (15.0%, 35/234). Escherichia coli (n = 118, 44.2%) was the most predominant bacteria, followed by Citrobacter freundii (n = 81, 30.3%). As much as 95.6% (392/410) of the isolates were penicillin-resistant, whereas only 36.2% (290/801) were carbapenem-resistant. A total of 96 (36.0%) MDR and 98 (36.7%) XDR Enterobacteriaceae were identified. Proteus mirabilis (44.4%, 8/18) predominated MDR cultures, whereas C. freundii (53.1%, 43/81) predominated XDR cultures. Multidrug resistant (38.4%, 71/154) and XDR Enterobacteriaceae (22.7%, 35/154) were chiefly uropathogens. Fluoroquinolone resistance rates in non-MDR, MDR, and XDR isolates were 19.9%, 63.2%, and 96.2%, respectively, whereas cephalosporin resistance rates were 28.6%, 72.9%, and 95.4% and penicillin resistance rates were 67.0%, 97.4%, and 98.0%. One-seventh of patients visiting the hospital were found to be infected with Enterobacteriaceae, and of these patients, at least one-fourth were infected with MDR strains.

Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Mobile genetic elements facilitate the transmission of antibiotic resistance genes in multidrug-resistant Enterobacteriaceae from duck farms

Multidrug-resistant (MDR) Enterobacteriaceae critically threaten duck farming and public health. The phenotypes, genotypes, and associated mobile genetic elements (MGEs) of MDR Enterobacteriaceae isolated from 6 duck farms in Zhejiang Province, China, were investigated. A total of 215 isolates were identified as Escherichia coli (64.65 %), Klebsiella pneumoniae (12.09 %), Proteus mirabilis (10.23 %), Salmonella (8.84 %), and Enterobacter cloacae (4.19 %). Meanwhile, all isolates were resistant to at least two antibiotics. Most isolates carried tet(A) (85.12 %), blaTEM (78.60 %) and sul1 (67.44 %) resistance genes. Gene co-occurrence analysis showed that the resistance genes were associated with IS26 and integrons. A conjugative IncFII plasmid pSDM004 containing all the above MGEs was detected in Proteus mirabilis isolate SDM004. This isolate was resistant to 18 antibiotics and carried the blaNDM-5 gene. MGEs, especially plasmids, are the primary antibiotic resistance gene (ARG) transmission route in duck farms. These findings provide a theoretical basis for the rational use of antibiotics in farms which are substantial for evaluating public health and food safety.

Asymptomatic gastrointestinal carriage of multidrug-resistant carbapenemase-producing Enterobacteriaceae among children under five years in a Kenyan hospital

ObjectivesAsymptomatic gastrointestinal carriage of carbapenem-resistant Enterobacteriaceae (CRE) is a threat to global health in developing countries with inadequate safe drinking water, poor hygiene, and weak antimicrobial stewardship; however, epidemiological data to guide CRE infection prevention and control is limited in these settings. We assessed asymptomatic CRE and carbapenem-producing Enterobacteriaceae (CPE) fecal carriage rates and associated risk factors among hospitalized children aged under 5 years. MethodsWe adopted a cross-sectional study at Mama Lucy Kibaki Hospital in Nairobi-City County, Kenya, between June and September 2022. We collected demographic and clinical characteristics using a structured questionnaire and clinical reports and analyzed stool/rectal swab samples by standard and automated bacteriological methods. ResultsAsymptomatic CRE and CPE fecal carriage rate was 2.25% (6/267), with six isolates recovered, predominated by Escherichia coli (33.33%) and Enterobacter cloacae subsp dissolvens (33.33%). Third-generation cephalosporin and ciprofloxacin resistance were highest in Citrobacter farmer and E. cloacae subsp cloacae. All CRE and CPE were multidrug-resistant, and except E. cloacae subsp cloacae, were 100% colistin-resistant. ConclusionsAsymptomatic gastrointestinal carriage of multidrug-resistant-CRE among hospitalized children under 5 years, presents a substantial public health threat. This calls for continuous surveillance including molecular characterization of isolates, to inform infection prevention and antimicrobial stewardship adherence in line with local and global plans on AMR.

Bdellovibrio bacteriovorus therapy, an emerging alternative to antibiotics

Abstract: The increase in multi-drug resistant (MDR) pathogens and the decline in the number of new antibiotics in the production pipeline pose a serious threat to our ability to treat infectious diseases. In this new landscape, once treatable diseases are now potentially life-threatening. This impending danger requires that urgent attention should be given to developing alternative strategies for combating MDR bacteria. A novel alternative is the use of predatory bacteria, B. bacteriovorus spp, that naturally prey on Gram-negative bacteria, including MDR Enterobacteriaceae. B. bacteriovorus has been shown to be non-pathogenic in animal models and on human cell lines, supporting its feasibility to be used to treat infections in animals and possibly humans. This document reviews various aspects of B. bacteriovorus biology, including its unique life cycle, "predatory toolbox", prey range, and recent research advances exploring B. bacteriovorus as an antimicrobial agent, stepping towards its use in human therapy. We also discuss the advantages and limitations of using B. bacteriovorus therapy and the strategies to overcome these limitations.

Whole genome sequencing-based cataloguing of antibiotic resistant genes in piggery waste borne samples

The growing use of antibiotics in livestock is one of the main causes of the rapid global spread of antimicrobial resistance (AMR). However, extensive research on AMR in animals is currently absent. In this article, we provide the bacterial antibiotic resistance genes (ARGs) from piggery waste samples in West Bengal, India, based on whole genome sequencing (WGS). According to the study, there are alarmingly high levels of Enterobacteriaceae in piggery waste, especially slaughterhouse waste, that are resistant to beta-lactam, aminoglycoside, sulphonamide, and tetracycline. We found several plasmids carrying multidrug-resistant Enterobacteriaceae including resistant to last-resort medications like colistin and carbapenems. Our findings will serve as a guide for developing AMR management policies for livestock in India and aid in understanding the current AMR profiles of pigs. To grasp the actual situation with AMR in the pig sector, large scale sample screening must be done.

In vitro synergy screens of FDA-approved drugs reveal novel zidovudine- and azithromycin-based combinations with last-line antibiotics against Klebsiella pneumoniae

Treatment of infections caused by multi-drug resistant (MDR) enterobacteria remains challenging due to the limited therapeutic options available. Drug repurposing could accelerate the development of new urgently needed successful interventions. This work aimed to identify and characterise novel drug combinations against Klebsiella pneumoniae based on the concepts of synergy and drug repurposing. We first performed a semi-qualitative high-throughput synergy screen (sHTSS) with tigecycline, colistin and fosfomycin (last-line antibiotics against MDR Enterobacteriaceae) against a FDA-library containing 1430 clinically approved drugs; a total of 109 compounds potentiated any of the last-line antibiotics. Selected hits were further validated by secondary checkerboard (CBA) and time-kill (TKA) assays, obtaining 15.09% and 65.85% confirmation rates, respectively. Accordingly, TKA were used for synergy classification based on determination of bactericidal activities at 8, 24 and 48 h, selecting 27 combinations against K. pneumoniae. Among them, zidovudine or azithromycin combinations with last-line antibiotics were further evaluated by TKA against a panel of 12 MDR/XDR K. pneumoniae strains, and their activities confronted with those clinical combinations currently used for MDR enterobacteria treatment; these combinations showed better bactericidal activities than usual treatments without added cytotoxicity. Our studies show that sHTSS paired to TKA are powerful tools for the identification and characterisation of novel synergistic drug combinations against K. pneumoniae. Further pre-clinical studies might support the translational potential of zidovudine- and azithromycin-based combinations for the treatment of these infections.

Stability and genetic insights of the co-existence of blaCTX-M-65, blaOXA-1, and mcr-1.1 harboring conjugative IncI2 plasmid isolated from a clinical extensively-drug resistant Escherichia coli ST744 in Shanghai.

Co-existence of colistin, β-lactam and carbapenem in multidrug-resistant Enterobacteriaceae isolates poses a serious threat to public health. In this study, we investigated and characterized the co-occurrence of blaCTX-M-65, blaOXA-1, and mcr-1.1 strain isolated from a clinical extensively-drug-resistant Escherichia coli ST744 in Shanghai. Antimicrobial susceptibility test was carried out by agar dilution methods. Whole genome sequencing was conducted, and resistance genes, and sequence types of colistin in E. coli isolates were analyzed. Plasmid stability and amino acid mutations were assessed in E. coli isolates. A colistin resistant E. coli ST744, named ECPX221, was identified out of 145 fecal samples collected. The strain carries a 60,168 IncI2 plasmid with the mcr-1.1 gene. The strain also has blaCTX-M-65, blaOXA-1, dfrA14, qnrS1, cmlA5, arr2, ampC, aph(4)-Ia, sul1, and aadA5 resistance genes. The plasmid pECPX221 was capable of conjugation with an efficiency of 2.6 × 10-2. Notably, 45% of the transconjugants were determined as mcr-1.1-harboring in the colistin-free environment after 60 generation of passage. No mutations occurred in pmrB, mgrB, and phoPQ gene in the mcr-1.1-harboring transconjugants. Bioinformatic analysis indicated pECPX221 shared highly similar backbone with the previously reported mcr-1.1-harboring pAH62-1, pMFDS1339.1, pSCZE4, and p2018-10-2CC. Furthermore, sequencing and phylogenetic analyses revealed a similarity between other MCR-1-homolog proteins, indicating that ECPX221 was colistin resistant. The stable transferable mcr-1.1-harboring plasmid found in the E. coli ST744 strain indicated the high risk to disseminate the extensively-drug-resistance phenotype among Enterobacteriaceae.

Frequency and Distribution of Multidrug Resistance Enterobacteriaceae Isolated from Hospital and Community Acquired Urinary Tract Infection Patient Attended at a Tertiary Level Care Hospital in Dhaka City

Background: Urinary tract infection is caused by different bacteria of family enterobacteriaceae in the hospital and community acquired infection.
 Objective: The purpose of the present study was to find out the bacteria causing urinary tract infection, frequency as well as their antibiotic resistance pattern.
 Methodology: This cross-sectional study was conducted in the Department of Microbiology at National Institute of Neurosciences and Hospital, Dhaka, Bangladesh from June 2017 to November 2017 for a period of six months. Different urine samples of patient who were presented with infection in the urinary tract were included as study population. Using sterile container urine samples were taken from both IPD and OPD patients of National Institute of Neurosciences and Hospital, Dhaka, Bangladesh. The urine specimen was inoculated on blood and MacConkey agar plates and incubated at 37°C for 24 hours. Identification was based on standard microbiological methods The antibiogram testing was done by using modified Kirby-Bauer method. 
 Results: A total of 200 urine samples were included for study of which 118(59%) cases showed positive and 82(41.0%) cases showed negative growth. Among 118 samples Escherichia coli were in 56(47.5%) isolates followed by Klebsiella pneumoniae were in 22(18.6%); Citrobacter freundii were in 20(16.9%); Pseudomonas species were 12(10.2%) and Proteus mirabilis were 8(6.8%). Escherichia coli was most predominant urinary tract infection causing species (47.5%) and found most resistant against cefixime (96.4%), azithromycin (92.9%), ciprofloxacin (71.4%); Klebsiella pneumonia was most resistant to amikacin, azithromycin (81.82%), ciprofloxacin, tazobactum/piperacillin (72.7%) cefixime, meropenem (63.7%). Citrobacter freundii was resistance to ciprofloxacin, cefixime (80.0%).
 Conclusion: Escherichia coli infections are more common in urinary tract infection patients and predominant resistant against cefixime followed by Klebsiella pneumonia.
 Bangladesh Journal of Infectious Diseases, December 2022;9(2):47-52

Whole-genome sequence analysis of carbapenem-resistant Enterobacteriaceae recovered from hospitalized patients

Carbapenems are among the few effective antibiotics against multidrug-resistant Enterobacteriaceae. This study aimed at characterizing the plasmid content and resistome of clinical carbapenem-resistant Enterobacteriaceae (CRE) recovered between 2016-2019 from hospitalized patients in Lebanon. Plasmid typing and whole-genome sequencing were used to study the genomic characteristics of 65 clinical CREs including 27 Escherichia coli, 24 Klebsiella pneumoniae, one Klebsiella quasipneumoniae, three Morganella morganii, three Citrobacter freundii, five Enterobacter hormaechei, and two Serratia marcescens. blaOXA-48 (33.8%; n=22) and blaOXA-48-like genes were among the detected resistance determinants, with two isolates co-harboring blaNDM-5. Various blaNDM variants, blaNDM-1 (16.9%; n=11), blaNDM-5 (9.2%; n=6), blaNDM-7 (9.2%; n=6), and blaNDM-19 (4.6%; n=3), different ESBLs and AmpC β-lactamases were detected. Carbapenem resistance determinants were linked a variety of incompatibility groups with IncFIB(K) (43.1%; n=28) being the most prevalent, followed by IncFIA (40.0%), IncL (35.4%), IncX3 (32.3%), IncI1 (32.3%), and IncFIIK (29.2%). We analyzed the clonality and resistance determinants of 65 multi-drug resistant (MDR) Enterobacteriaceae recovered in the period between 2016-2019 from a large tertiary hospital in Lebanon. NDM variants, OXA-48 and OXA-181were the most prevalent detected carbapenemases and were mostly linked to the dissemination of IncL, IncX3, and IncF. This study reinforces the need to track the spread and dominance of clinically relevant carbapenemase-encoding plasmids in healthcare settings.

Rats as sources of multidrug-resistant Enterobacteriaceae in animal production environments.

Rattus norvegicus and Rattus rattus are commensal pest rodents, considered reservoirs and vectors of zoonotic pathogens. In livestock farms, the wide use of antimicrobials and their release into the environment lead to high long-term residual concentrations, which may in turn lead to the occurrence of antimicrobial resistance (AMR). Farm environments serve as AMR sources, resulting in the transmission of antimicrobial-resistant bacteria and their AMR genes of livestock origin into wildlife. This study aimed to analyse the profile of enterobacteria carrying AMR determinants in rats captured in livestock farms to determine their potential vectors as for the spread of AMR. To this end, 56 rats (52 R. norvegicus and 4 R. rattus) were live-trapped on 11 farms (pig, dairy, poultry and mixed farms) located in central Argentina, from spring 2016 to autumn 2017. From 50 of the R. norvegicus individuals and three of the R. rattus individuals found in 10 of the farms, we isolated 53 Escherichia coli and five Salmonella strains. Susceptibility to antimicrobials, genotypic profiles, minimal inhibitory concentration of colistin and the presence of mcr-1 and genes encoding extended-spectrum β-lactamase (ESBL) were determined. Of the 58 isolates not susceptible to different antimicrobial classes, 28 of the E. coli strains and two of the Salmonella strains were defined as multi-drug resistant (MDR). S. Westhampton and S. Newport recovered were not susceptible to ampicillin or all the cephems tested. One of the E. coli obtained showed resistance to colistin and harboured the mcr-1 gene, demonstrated by PCR and conjugation. In two ESBL-producing Salmonella isolated from rats, CTX-M-2 genes were responsible for the observed resistance to third-generation cephalosporins. The MDR E. coli isolates showed several different resistance patterns (23), although some of them were the same in different individuals and different farms, with six resistance patterns, evidencing the dispersion of strains. These findings suggest that rats play a role in the dissemination of AMR determinants between animal, humans and environmental reservoirs.

Microplastics accumulate priority antibiotic-resistant pathogens: Evidence from the riverine plastisphere

Microplastics (MPs) might accumulate and transport antibiotic-resistant bacteria (ARB) in aquatic systems. We determined the abundance and diversity of culturable ciprofloxacin- and cefotaxime-resistant bacteria in biofilms covering MPs placed in river water, and characterized priority pathogens from these biofilms. Our results showed that the abundance of ARB colonizing MPs tends to be higher compared to sand particles. Also, higher numbers were cultivated from a mixture of polypropylene (PP), polyethylene (PE) and polyethylene terephthalate (PET), compared to PP and PET alone. Aeromonas and Pseudomonas isolates were the most frequently retrieved from MPs placed before a WWTP discharge while Enterobacteriaceae dominated the culturable plastisphere 200 m after the WWTP discharge. Ciprofloxacin- and/or cefotaxime-resistant Enterobacteriaceae (n = 54 unique isolates) were identified as Escherichia coli (n = 37), Klebsiella pneumoniae (n = 3), Citrobacter spp. (n = 9), Enterobacter spp. (n = 4) and Shigella sp. (n = 1). All isolates presented at least one of the virulence features tested (i.e. biofilm formation, haemolytic activity and production of siderophores), 70% carried the intI1 gene and 85% exhibited a multi-drug resistance phenotype. Plasmid-mediated quinolone resistance genes were detected in ciprofloxacin-resistant Enterobacteriaceae [aacA4-cr (40% of the isolates), qnrS (30%), qnrB (25%), and qnrVC (8%)], along with mutations in gyrA (70%) and parC (72%). Cefotaxime-resistant strains (n = 23) harbored blaCTX-M (70%), blaTEM (61%) and blaSHV (39%). Among CTX-M producers, high-risk clones of E. coli (e.g. ST10 or ST131) and K. pneumoniae (ST17) were identified, most of which carrying blaCTX-M-15. Ten out of 16 CTX-M producers were able to transfer blaCTX-M to a recipient strain. Our results demonstrated the occurrence of multidrug resistant Enterobacteriaceae in the riverine plastisphere, harboring ARGs of clinical concern and exhibiting virulence traits, suggesting a contribution of MPs to the dissemination of antibiotic-resistant priority pathogens. The type of MPs and especially water contamination (e.g. by WWTPs discharges) seem to determine the resistome of the riverine plastisphere.

The Detection of Fosfomycin-Modifying Enzymes (fos) in Uropathogenic Enterobacterale, Azerbaijan, Iran.

Enterobacteriaceae is the most common agent of urinary tract infections (UTIs). Multidrug resistant (MDR) and XDR (extensively drug-resistant) Enterobacteriaceae in UTIs have increased in the world. The present study aimed to study the fosfomycin resistance frequency and the fosfomycin resistance genes among Enterobacteriaceae isolated from UTIs. The urine was collected and cultured in the standard protocol. To determine the susceptibility testing to fosfomycin in 211 isolates, agar dilution and disk agar diffusion methods were used. MDR was nonsusceptibility to at least one agent in three or more antimicrobial categories. The fosfomycin resistance genes were also evaluated by PCR. The frequency of resistance to fosfomycin was in 14 (6.6%) and 15 (7.1%) isolates by the disk agar diffusion and MIC assays, respectively. However, the MIC50 and MIC90 existed at 8 μg/mL and 16 μg/mL, respectively. The MDR was found in 80%. The frequencies of fosfomycin resistance genes were 5 (33.3%), 3 (20%), 2 (13.3%), 1 (6.6%), and 1 (6.6%) for fosC, fosX, fosA3, fosA, and fosB2, respectively. The fosB and fosC2 were not found. A low resistance rate to fosfomycin is observed. Fosfomycin is still one of the most effective and valuable alternative antibiotics against MDR Enterobacteriaceae isolated from UTIs in our region.