Phenotypic and Genotypic Characteristics of Antimicrobial Resistance in Citrobacter freundii Isolated from Domestic Ducks (Anas platyrhynchos domesticus) in Bangladesh.

Antimicrobial resistance (AMR) in Citrobacter freundii poses a serious challenge as this species is one of the sources of nosocomial infection and causes diarrheal infections in humans. Ducks could be the potential source of multidrug-resistant (MDR) C. freundii; however, AMR profiles in C. freundii from non-human sources in Bangladesh have remained elusive. This study aimed to detect C. freundii in domestic ducks (Anas platyrhynchos domesticus) in Bangladesh and to determine their phenotypic and genotypic antibiotic susceptibility patterns. A total of 150 cloacal swabs of diseased domestic ducks were screened using culturing, staining, biochemical, polymerase chain reaction (PCR), and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) to detect C. freundii. Phenotypic and genotypic antibiotic susceptibility patterns were done by the disk diffusion method and PCR, respectively. In total, 16.67% (25/150) of the samples were positive for C. freundii. C. freundii isolates showed a range of 20% to 96% resistance to cefotaxime, gentamicin, levofloxacin, ciprofloxacin, cotrimoxazole, tetracycline, ampicillin, and cephalexin. More than 60% of the isolates were phenotypically MDR, and the index of multiple antibiotic resistance ranged from 0.07 to 0.79. Genes encoding resistance to beta-lactams [blaTEM-1-88% (22/25), blaCMY-2-56% (14/25), blaCMY-9-8% (2/25), and blaCTX-M-14-20% (5/25)], sulfonamides [sul1-52% (13/25), sul2-24% (6/25)], tetracyclines [tetA-32% (8/25) and tetB-4% (1/25)], aminoglycosides [aacC4-16% (4/25)], and fluoroquinolones [qnrA-4% (1/25), qnrB-12% (3/25), and qnrS-4% (1/25)] were detected in the isolated C. freundii. To the best of our knowledge, this is the first study in Bangladesh to detect MDR C. freundii with their associated resistance genes from duck samples. We suggest addressing the burden of diseases in ducks and humans and associated AMR issues using the One Health approach.

Isothermal microcalorimetry vs checkerboard assay to evaluate in-vitro synergism of meropenem-amikacin and meropenem-colistin combinations against multi-drug-resistant Gram-negative pathogens.

OBJECTIVES: To evaluate the activity of meropenem-amikacin and meropenem-colistin combinations with checkerboard broth microdilution (CKBM) compared with isothermal microcalorimetry (ITMC) assays against a multi-centric collection of multi-drug-resistant Gram-negative clinical isolates; and to compare the fractional inhibitory concentration (FIC) index and time to results of CKBM and ITMC. METHODS: A collection of 333 multi-drug-resistant Gram-negative clinical isolates showing reduced susceptibility to meropenem (121 Klebsiella pneumoniae, 14 Escherichia coli, 130 Pseudomonas aeruginosa and 68 Acinetobacter baumannii) isolated from different centres (Florence, Madrid, Rotterdam and Stockholm) was included in the study. The antimicrobial activity of meropenem-amikacin and meropenem-colistin combinations was evaluated with CKBM and ITMC. FIC index results were interpreted as synergistic/additive and indifferent for values ≤0.5/0.51, respectively. Whole-genome sequencing data of a subset of strains were used to evaluate their clonality. RESULTS: In total, 254 and 286 strains were tested with meropenem-colistin and meropenem-amikacin combinations with ITMC and CKBM, respectively. Synergistic/additive effects were observed for 46 strains (20 K. pneumoniae, four E. coli, 22 P. aeruginosa) and 20 strains (three K. pneumoniae, 11 P. aeruginosa and six A. baumannii) with meropenem-amikacin and meropenem-colistin combinations, respectively, with CKBM. ITMC showed good concordance with CKBM, with 89.5% and 92.2% of cases interpreted within the same FIC index category for meropenem-amikacin and meropenem-colistin combinations, respectively. Most of the synergistic/additive effects were detected within 6 h by ITMC. CONCLUSIONS: ITMC showed very good concordance with CKBM against a large collection of multi-drug-resistant Gram-negative clinical isolates, and could be implemented for the rapid evaluation of in-vitro activity of antimicrobial combinations.

Genomic Epidemiology of Carbapenemase-Producing and Colistin-Resistant Enterobacteriaceae among Sepsis Patients in Ethiopia: a Whole-Genome Analysis.

Sepsis due to carbapenemase-producing and colistin-resistant Enterobacteriaceae is a global health threat. A multicenter study was conducted between October 2019 and September 2020 at four hospitals located in different parts of Ethiopia. From a total of 1,416 sepsis patients, blood culture was performed. Enterobacteriaceae were confirmed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Carbapenem and colistin susceptibility testing was performed using disk diffusion, broth microdilution, and Etest strip. Enterobacteriaceae isolates (n = 301) were subjected to whole-genome sequencing using Illumina HiSeq 2500. SPAdes version 3.9 was used for genome assembly. Carbapenem and colistin resistance genes, chromosomal point mutations, sequence types, and plasmid replicons were identified using tools at the Center for Genomic Epidemiology. Phylogeny structure was constructed using CSI Phylogeny 1.4. Visualization of trees and metadata was done using iTOL v6.5.2. Among 301 Enterobacteriaceae, 22 Klebsiella pneumoniae, 2 Klebsiella variicola, and 3 Enterobacter cloacae isolates showed reduced susceptibility to meropenem (7% of tested isolates). blaNDM-1, blaNDM-5, and blaOXA-181 were variants of carbapenemase genes detected. Co-occurrence of blaNDM-5 and blaOXA-181 was detected with 4 K. pneumoniae strains. K. pneumoniae and K. variicola showed chromosomal alterations of ompK36 and ompk37. Plasmid incompatibility (Inc) groups Col, IncC, IncHI, IncF, IncFII, IncR, and IncX3 were identified among carbapenem-resistant K. pneumoniae and E. cloacae isolates. Two mcr-9 genes were detected from Salmonella species and K. pneumoniae. The dissemination of carbapenemase-producing Enterobacteriaceae in all hospitals is worrying. Multiple carbapenemase genes were detected, with blaNDM variants the most frequent. The occurrence of colistin-resistant Enterobacteriaceae among sepsis patients is critical. Implementation of effective antimicrobial stewardship is urgently needed.

Molecular Characterization of Clinically Relevant Extended-Spectrum ß-Lactamases blaCTX-M-15-Producing Enterobacteriaceae Isolated from Free-Range Chicken from Households in Bangladesh.

The study explored the potential colonization and characterization of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in the gut of free-range poultry from the rural households in Bangladesh. From 48 households located in several rural regions (eastern, western, and southern) of Bangladesh, 180 poultry fecal samples were collected to isolate ESBL-producing Enterobacteriaceae. ESBL producers were characterized by susceptibility testing, conjugation experiment, conventional polymerase chain reactions (PCRs), and multilocus sequence typing (MLST) followed by sequencing. Total 23% (42/180) poultry were ESBL positive consisting of Escherichia coli (n = 41) and Klebsiella pneumoniae (n = 1). ESBL producers were resistant to Cefotaxime (CTX; 100%), Cefepime (100%), Amoxicillin-clavulanic acid (36%), Ciprofloxacin (31%), and Trimethoprim-sulfamethoxazole (24%), and 12% isolates were multidrug resistant. All ESBL producers were carrying blaCTX-M-15-like genotype.Isolates were also carrying genes for quinolone resistance [qnrS1, aac(6')-Ib-cr], silver resistance (silE), and mercury resistance (merA). Isolates were negative for 025b-ST131 clone, mcr-1, and blaOXA-48 gene. The repetitive element PCR revealed 15 different clones of E. coli and some of these clones were found to be common in 3 sampling locations. MLST analysis of E. coli revealed 9 different sequence types (STs); ST4, ST156, ST542, ST1140, ST1290, ST4628, ST5114, ST9768, and ST11317. ESBL producers were carrying transferable plasmids and 4 different plasmid replicon types; IncI1 (29%), IncY (7%), IncFIB (7%), and IncF1A (5%). The findings from the study confirmed that free-range poultry are potential ESBL carriers with coresistance to other antibiotic classes, metals, and biocides. This study confirms that free-range poultry in Bangladesh living close to humans without any direct antibiotic exposure could carry ESBL bacteria. Free-range poultry could be reservoir as well as a potential spreader of pathogenic E. coli and antibiotic- or biocide-resistant genes.

Sepsis: emerging pathogens and antimicrobial resistance in Ethiopian referral hospitals.

BACKGROUND: Sepsis due to multidrug resistant (MDR) bacteria is a growing public health problem mainly in low-income countries. METHODS: A multicenter study was conducted between October 2019 and September 2020 at four hospitals located in central (Tikur Anbessa and Yekatit 12), southern (Hawassa) and northern (Dessie) parts of Ethiopia. A total of 1416 patients clinically investigated for sepsis were enrolled. The number of patients from Tikur Anbessa, Yekatit 12, Dessie and Hawassa hospital was 501, 298, 301 and 316, respectively. At each study site, blood culture was performed from all patients and positive cultures were characterized by their colony characteristics, gram stain and conventional biochemical tests. Each bacterial species was confirmed using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI TOF). Antimicrobial resistance pattern of bacteria was determined by disc diffusion. Logistic regression analysis was used to assess associations of dependent and independent variables. A p-value < 0.05 was considered as statistically significant. The data was analyzed using SPSS version 25. RESULTS: Among 1416 blood cultures performed, 40.6% yielded growth. Among these, 27.2%, 0.3% and 13.1%, were positive for pathogenic bacteria, yeast cells and possible contaminants respectively. Klebsiella pneumoniae (26.1%), Klebsiella variicola (18.1%) and E. coli (12.4%) were the most frequent. Most K. variicola were detected at Dessie (61%) and Hawassa (36.4%). Almost all Pantoea dispersa (95.2%) were isolated at Dessie. Rare isolates (0.5% or 0.2% each) included Leclercia adecarboxylata, Raoultella ornithinolytica, Stenotrophomonas maltophilia, Achromobacter xylosoxidans, Burkholderia cepacia, Kosakonia cowanii and Lelliottia amnigena. Enterobacteriaceae most often showed resistance to ampicillin (96.2%), ceftriaxone (78.3%), cefotaxime (78%), cefuroxime (78%) and ceftazidime (76.4%). MDR frequency of Enterobacteriaceae at Hawassa, Tikur Anbessa, Yekatit 12 and Dessie hospital was 95.1%, 93.2%, 87.3% and 67.7%, respectively. Carbapenem resistance was detected in 17.1% of K. pneumoniae (n = 111), 27.7% of E. cloacae (n = 22) and 58.8% of Acinetobacter baumannii (n = 34). CONCLUSION: Diverse and emerging gram-negative bacterial etiologies of sepsis were identified. High multidrug resistance frequency was detected. Both on sepsis etiology types and MDR frequencies, substantial variation between hospitals was determined. Strategies to control MDR should be adapted to specific hospitals. Standard bacteriological services capable of monitoring emerging drug-resistant sepsis etiologies are essential for effective antimicrobial stewardship.

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

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

Molecular Epidemiology of Extended-Spectrum Beta-Lactamase and AmpC Producing Enterobacteriaceae among Sepsis Patients in Ethiopia: A Prospective Multicenter Study.

Extended-spectrum beta-lactamases (ESBLs) and AmpC producing Enterobacteriaceae are public health threats. This study aims to characterize ESBL and AmpC producing Enterobacteriaceae isolated from sepsis patients. A multicenter study was conducted at four hospitals located in central (Tikur Anbessa and Yekatit 12), southern (Hawassa) and northern (Dessie) parts of Ethiopia. Blood culture was performed among 1416 sepsis patients. Enterobacteriaceae (n = 301) were confirmed using MALDI-TOF and subjected for whole genome sequencing using the Illumina (HiSeq 2500) system. The overall genotypic frequencies of ESBL and AmpC producing Enterobacteriaceae were 75.5% and 14%, respectively. The detection of ESBL producing Enterobacteriaceae at Hawassa, Yekatit 12, Tikur Anbessa and Dessie was 95%, 90%, 82% and 55.8%, respectively. The detection frequency of blaCTX-M, blaTEM and blaSHV genes was 73%, 63% and 33%, respectively. The most frequently detected ESBL gene was blaCTX-M-15 (70.4%). The common AmpC genes were blaACT (n = 22) and blaCMY (n = 13). Of Enterobacteriaceae that harbored AmpC (n = 42), 71% were ESBL co-producers. Both blaTEM-1B (61.5%) and blaSHV-187 (27.6%) were the most frequently detected variants of blaTEM and blaSHV, respectively. The molecular epidemiology of ESBL producing Enterobacteriaceae showed high frequencies and several variants of ESBL and AmpC genes. Good antimicrobial stewardship and standard bacteriological laboratory services are necessary for the effective treatment of ESBL producing Enterobacteriaceae.

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.

Effective antimicrobial combination in vivo treatment predicted with microcalorimetry screening.

OBJECTIVES: The worldwide emergence of antibiotic resistance calls for effective exploitation of existing antibiotics. Antibiotic combinations with different modes of action can synergize for successful treatment. In the present study, we used microcalorimetry screening to identify synergistic combination treatments against clinical MDR isolates. The synergistic effects were validated in a murine infection model. METHODS: The synergy of meropenem combined with colistin, rifampicin or amikacin was tested on 12 isolates (1 Escherichia coli, 5 Klebsiella pneumoniae, 3 Pseudomonas aeruginosa and 3 Acinetobacter baumannii) in an isothermal microcalorimeter measuring metabolic activity. One A. baumannii strain was tested with two individual pairings of antibiotic combinations. The microcalorimetric data were used to predict in vivo efficacy in a murine peritonitis/sepsis model. NMRI mice were inoculated intraperitoneally and after 1 h treated with saline, drug X, drug Y or X+Y. Bacterial load was determined by cfu in peritoneal fluid and blood after 4 h. RESULTS: In vitro, of the 13 combinations tested on the 12 strains, 3 of them exhibited a synergistic reduction in MIC (23% n = 3/13), 5 showed an additive effect (38.5% n = 5/13) and 5 had indifferent or antagonistic effects (38.5% n = 5/13). There was a significant correlation (P = 0.024) between microcalorimetry-screening FIC index values and the log reduction in peritoneal fluid from mice that underwent combination treatment compared with the most effective mono treatment. No such correlation could be found between chequerboard and in vivo results (P = 0.16). CONCLUSIONS: These data support microcalorimetic metabolic readout to predict additive or synergistic effects of combination treatment of MDR infections within hours.

Extended-Spectrum-ß-Lactamase- and Plasmid AmpC-Producing Escherichia coli Causing Community-Onset Bloodstream Infection: Association of Bacterial Clones and Virulence Genes with Septic Shock, Source of Infection, and Recurrence.

Invasive infections due to extended-spectrum-ß-lactamase- and pAmpC-producing Escherichia coli (ESBL/pAmpC-EC) are an important cause of morbidity, often caused by the high-risk clone sequence type (ST131) and isolates classified as extraintestinal pathogenic E. coli (ExPEC). The relative influence of host immunocompetence versus microbiological virulence factors in the acquisition and outcome of bloodstream infections (BSI) is poorly understood. Herein, we used whole-genome sequencing on 278 blood culture isolates of ESBL/pAmpC-EC from 260 patients with community-onset BSI collected from 2012 to 2015 in Stockholm to study the association of virulence genes, sequence types, and antimicrobial resistance with severity of disease, infection source, ESBL/pAmpC-EC BSI low-risk patients, and patients with repeated episodes. ST131 subclade C2 comprised 29% of all patients. Factors associated with septic shock in multivariable analysis were patient host factors (hematologic cancer or transplantation and reduced daily living activity), presence of the E. coli virulence factor iss (increased serum survival), absence of phenotypic multidrug resistance, and absence of the genes pap and hsp Adhesins, particularly pap, were associated with urinary tract infection (UTI) source, while isolates from post-prostate biopsy sepsis had a low overall number of virulence operons, including adhesins, and commonly belonged to ST131 clades A, B, and subclade C1, ST1193, and ST648. ST131 was associated with recurrent episodes. In conclusion, the most interesting finding is the association of iss with septic shock. Adhesins are important for UTI pathogenesis, while otherwise low-pathogenic isolates from the microbiota can cause post-prostate biopsy sepsis.

Fecal Carriage of Extended-Spectrum ß-Lactamases in Healthy Humans, Poultry, and Wild Birds in León, Nicaragua-A Shared Pool of blaCTX-M Genes and Possible Interspecies Clonal Spread of Extended-Spectrum ß-Lactamases-Producing Escherichia coli.

Antibiotic-resistant bacteria are a major concern in the healthcare of today, especially the increasing number of gram-negative bacteria producing ß-lactamases such as extended-spectrum ß-lactamases (ESBLs). However, little is known about the relationship of ESBL producers in humans and domestic and wild birds, especially in a low-income setting. Therefore, we studied the fecal carriage of ESBL-producing Escherichia coli and Klebsiella pneumoniae in healthy humans, poultry, and wild birds in the vicinity of León, Nicaragua. Three hundred fecal samples were collected during December 2012 from humans (n = 100), poultry (n = 100) and wild birds (n = 100). The samples were examined for ESBL-producing E. coli and K. pneumoniae, revealing the prevalence of 27% in humans, 13% in poultry, and 8% in wild birds. Further characterization of the ESBL-producing isolates was performed through polymerase chain reaction (PCR) (NDM, CTX-M), epidemiological typing (ERIC2-PCR), multilocus sequence typing, and sequencing. ESBL producers harbored blaCTX-M-2, blaCTX-M-15, blaCTX-M-22, and blaCTX-M-3 genotypes. The blaCTX-M-15 constituted the absolute majority of ESBL genes among all samples. ERIC-PCR demonstrated highly related E. coli clones among humans, poultry, and wild birds. Clinically relevant E. coli clone ST648 was found in humans and poultry. There is a shared pool of blaCTX-M genes between humans and domesticated and wild birds in Nicaragua, and the results suggest shared clones of ESBL-producing E. coli. The study adds to the notion that wild birds and poultry can pick up antibiotic-resistant bacteria of human origin and function as a melting pot of resistance. Structured surveillance programs of antimicrobial resistance and a more regulated prescription of antibiotics are warranted in Nicaragua.

Occurrence of Hybrid Escherichia coli Strains Carrying Shiga Toxin and Heat-Stable Toxin in Livestock of Bangladesh.

Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) are important causes of diarrhea in humans and animals worldwide. Although ruminant animals are the main source of STEC, diarrhea due to this pathotype is very low in Bangladesh where ETEC remains the predominant group associated with childhood diarrhea. In the present study, E. coli strains (n = 35) isolated from Bangladesh livestock (goats, sheep, and cattle) and poultry (chicken and ducks) were analyzed for the presence of major virulence factors, such as Shiga toxins (STX-1 and STX-2), heat-labile toxin, and heat-stable toxins (STa and STb). Multiplex polymerase chain reaction results revealed 23 (66%) E. coli strains to be virulent possessing either sta (n = 5), stx (stx1, n = 8; stx2, n = 2), or both (n = 8) genes in varying combinations. Thirty-four percent (8/23) of strains from livestock were hybrid type that carried both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene sta. Serotyping results revealed that the ETEC strains belonged to five serotypes, namely O36:H5, O174:H-, O152:H8, O109:H51, and O8:H21, while the STEC-producing strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16 (n = 2), OR:H2 (n = 1), O110:H16 (n = 1), and O152:H8 (n = 1). The STEC-ETEC hybrid strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16, OR:H2, and O152:H8. Forty percent (2/5) of the ETEC and 20% (2/10) of the STEC strains were multidrug resistant with the highest drug resistance (50%) being found in the hybrid strains. Molecular fingerprinting determined by pulsed-field gel electrophoresis and cluster analyses by dendrogram revealed that, genetically, STEC-ETEC hybrid strains were highly heterogeneous. Multidrug-resistant E. coli STEC-ETEC hybrid strains in domesticated animals pose a public health threat for humans in Bangladesh.

Absence of vancomycin-resistant enterococci among highly ESBL-positive crows (Corvus splendens) foraging on hospital waste in Bangladesh.

BACKGROUND: Vancomycin-resistant enterococci (VRE) have emerged as a growing problem in hospitals; however, domesticated animals, poultry, and wild birds are acting as potential reservoirs. There is a knowledge gap in the Epidemiology of VRE from Bangladesh. METHODS: To study the prevalence of VRE and the mechanisms of resistance implicated among wild birds, 238 fecal samples were collected in 2010 from house crows (Corvus splendens) foraging on hospital waste in Bangladesh. Fecal samples were screened by analyzing color change in broth and screening for vanA and vanB resistant genes by PCR. RESULTS: Neither vanA nor vanB genes were detected from the fecal samples. The house crow does not seem to constitute a reservoir for VRE. CONCLUSION: The zero prevalence is an indication that foraging on hospital waste does not constitute a major risk of VRE carriage in house crows and this is the first study to focus on the prevalence of VRE from wild birds in Bangladesh.

Mallard or chicken? Comparing the isolation of avian influenza A viruses in embryonated Mallard and chicken eggs.

BACKGROUND: To date, the most efficient and robust method for isolating avian influenza A viruses (IAVs) is using embryonated chicken eggs (ECEs). It is known that low-pathogenic avian IAVs undergo rapid genetic changes when introduced to poultry holdings, but the factors driving mutagenesis are not well understood. Despite this, there is limited data on the effects of the standard method of virus isolation of avian-derived viruses, that is, whether isolation in ECEs causes adaptive changes in avian IAVs. Eggs from a homologous species could potentially offer an isolation vessel less prone to induce adaptive changes. METHODS: We performed eight serial passages of two avian IAVs isolated from fecal samples of wild Mallards in both ECEs and embryonated Mallard eggs, and hemagglutination assay titers and hemagglutinin sequences were compared. RESULTS: There was no obvious difference in titers between ECEs and embryonated Mallard eggs. Sequence analyses of the isolates showed no apparent difference in the rate of introduction of amino acid substitutions in the hemagglutinin gene (three substitutions in total in embryonated Mallard eggs and two substitutions in ECEs). CONCLUSION: Embryonated Mallard eggs seem to be good isolation vessels for avian IAVs but carry some practical problems such as limited availability and short egg-laying season of Mallards. Our study finds isolation of Mallard-derived avian IAVs in ECEs non-inferior to isolation in embryonated Mallard eggs, but more research in the area may be warranted as this is a small-scale study.

Antimicrobial-resistant and ESBL-producing Escherichia coli in different ecological niches in Bangladesh.

INTRODUCTION: The rapid and wide-scale environmental spread of multidrug-resistant bacteria in different ecosystems has become a serious issue in recent years. OBJECTIVES: To investigate the epidemiology of antimicrobial resistance and extended spectrum beta-lactamase (ESBL) in Bangladeshi wild birds and aquatic environments, samples were taken from Open Bill Stork (Anastomus oscitans) (OBS) and the nearby water sources. METHODS: Water and fresh fecal samples were collected from several locations. All samples were processed and cultured for Escherichia coli and tested for antibiotic susceptibility against commonly used antibiotics. ESBL producers were characterized at genotypic level using polymerase chain reaction (PCR), sequencing, multilocus sequence typing, and rep-PCR. RESULTS AND DISCUSSION: A total of 76 E. coli isolates from the 170 OBS and 8 E. coli isolates from three river sources were isolated. In total, 29% of E. coli isolated from OBS and all of the E. coli isolated from water sources were resistant to at least one of the tested antimicrobials. Resistant phenotypes were observed with all antimicrobials except tigecycline, gentamicin, imipenem, and chloramphenicol. Multidrug resistance was observed in 2.6% of OBS and 37.5% of the water isolates. Also, 1.2% of the ESBL-producing E. coli were isolated from OBS, whereas 50% of the E. coli isolated from water sources were ESBL producers possessing the CTX-M-15 gene. The most concerning aspect of our findings was the presence of human-associated E. coli sequence types in the water samples, for example, ST156-complex156, ST10-complex10 and ST46. CONCLUSION: This study reports the presence of multidrug-resistant ESBL-producing E. coli in OBSs and nearby aquatic sources in Bangladesh.

Zero prevalence of vancomycin-resistant enterococci among Swedish preschool children.

OBJECTIVES: Enterococci are a natural part of the bacterial flora of humans, animals, and insects and are frequently found in the community. Vancomycin-resistant enterococci (VRE) have emerged as a growing problem, associated with high morbidity and mortality. The aim of this study was to investigate the prevalence of VRE among healthy Swedish preschool children and ascertain whether they constitute a reservoir for the bacteria. METHODS: In total, 313 individual diapers were collected from preschools in Uppsala, Sweden. Fecal samples were screened by analyzing the color change in a broth followed by polymerase chain reaction for vanA and vanB genes, which are associated with vancomycin resistance. RESULTS: Neither vanA nor vanB genes could be detected from the samples. CONCLUSIONS: Preschool children in Uppsala do not constitute a reservoir for VRE. The zero prevalence is consistent with the overall decline in VRE prevalence in Sweden during the last years.

The gull (Chroicocephalus brunnicephalus) as an environmental bioindicator and reservoir for antibiotic resistance on the coastlines of the Bay of Bengal.

The presence and frequency of multiresistant bacteria in wild birds act as indicators of the environmental contamination of antibiotic resistance. To explore the rate of contamination mediated by Escherichia coli, 150 fecal samples from the brown-headed gull (Chroicocephalus brunnicephalus) and 8 water samples from the Bay of Bengal area were collected, cultured, and tested for antibiotic susceptibility. Special attention was paid to extended-spectrum beta-lactamase (ESBL)-producing isolates, which were further characterized genetically. Antibiotic resistance was found in 42.3% (36/85) of the E. coli isolates and multidrug resistance in 11.8%. Isolates from the area with a higher human activity were more resistant than those from an area with a lower level of activity. Most frequent was resistance to ampicillin (29.4%), followed by trimethoprim-sulfamethoxazole (24.7%) and quinolones (22.4%). Carriage of ESBL-producing E. coli was relatively high (17.3%) in the gulls, whereas no ESBL producers were found in the water. All ESBL-producing E. coli isolates, but one, carried bla(CTX-M-15) or bla(CTX-M-15)-like genes. A bla(CTX-M-14)-like enzyme was found as an exception. Gulls from two different colonies shared E. coli clones and harbored the clinically relevant sequence types ST10, ST48, and ST131. The high frequency of antibiotic resistance and ESBL production among E. coli isolates from gulls indicates that the environmental contamination of antibiotic resistance has already gone far on the coastlines of the Bay of Bengal. Considering the limited control over the antibiotic consumption and waste from human activities in Bangladesh, there is no easy solution in sight.

Handling and welfare of bovine livestock at local abattoirs in Bangladesh.

The World Organization for Animal Health (OIE) allows rope casting and the tying of legs for nonhuman animal slaughter without stunning. The handling and welfare of bovine livestock (Bos indicus and Bubalus bubalis) were studied in 8 local abattoirs in 5 districts of Bangladesh. A total of 302 animals were evaluated. At the local abattoirs, approximately 1/3 of the cattle and water buffalo were either emaciated or injured/sick. The size and vigor of the animals determined the casting method. Small and weak animals were cast on concrete floors by lifting a foreleg followed by pushing, or simply by twisting the head of the animal and then binding the legs with rope. Vigorous animals such as buffalo were cast using ropes and human force. Bleeding was slow and flaying was sometimes initiated before the animals were unconscious. Pulling and tearing of the trachea and pouring of water into the exposed trachea shortly after cutting were also observed in some cases. The overall animal handling was unnecessarily rough and the OIE standards were not implemented. Animals are subjected to considerable mistreatment, and there is an urgent need for the training and education of the staff in abattoirs concerning humane slaughtering practices as well as a need to build modern slaughtering plants in Bangladesh.

Antibiotic resistance patterns in Escherichia coli from gulls in nine European countries.

BACKGROUND: The prevalence of antibiotic resistant faecal indicator bacteria from humans and food production animals has increased over the last decades. In Europe, resistance levels in Escherichia coli from these sources show a south-to-north gradient, with more widespread resistance in the Mediterranean region compared to northern Europe. Recent studies show that resistance levels can be high also in wildlife, but it is unknown to what extent resistance levels in nature conform to the patterns observed in human-associated bacteria. METHODS: To test this, we collected 3,158 faecal samples from breeding gulls (Larus sp.) from nine European countries and tested 2,210 randomly isolated E. coli for resistance against 10 antibiotics commonly used in human and veterinary medicine. RESULTS: Overall, 31.5% of the gull E. coli isolates were resistant to ≥1 antibiotic, but with considerable variation between countries: highest levels of isolates resistant to ≥1 antibiotic were observed in Spain (61.2%) and lowest levels in Denmark (8.3%). For each tested antibiotic, the Iberian countries were either the countries with the highest levels or in the upper range in between-country comparisons, while northern countries generally had a lower proportion of resistant E. coli isolates, thereby resembling the gradient of resistance seen in human and food animal sources. CONCLUSION: We propose that gulls may serve as a sentinel of environmental levels of antibiotic resistant E. coli to complement studies of human-associated microbiota.