Multiple Antimicrobial Resistance Research Articles

Detection and antibiogram profile of diarrheagenic Escherichia coli isolated from two abattoir settings in northwest Ethiopia: a one health perspective

BackgroundDiarrheagenic Escherichia coli (E. coli) is a zoonotic pathogen that contaminates abattoir workers, slaughter environments, slaughter equipment, and carcasses during abattoir processing. Infection with E. coli is associated with the consumption of contaminated food and water, and it is a potential threat to the health and welfare of both humans and animals. Hence, this study aimed to detect diarrheagenic E. coli and assess its antibiogram profile in two abattoir settings, in one health lens.MethodsA cross-sectional study in one health approach was conducted from December 2020 to June 2021. A total of 384 samples from abattoir workers’ hands, carcasses, knives, cattle feces, abattoir water and effluents were collected. Bacterial culture and biochemical tests were conducted to isolate E. coli, while conventional polymerase chain reaction was performed to identify virulence genes. The antibiogram of diarrheagenic E. coli was tested against nine antimicrobials using the Kirby Bauer disk diffusion method.ResultsA total of 115 (29.95%) E. coli were isolated from the 384 samples, and from these isolates, about 17 (14.8%) were confirmed to be diarrheagenic E. coli (DEC). Among the DEC pathotypes, nine (52.94%), five (29.4%), and three (17.65%) were Shiga toxin-producing, enterohemorrhagic, and enterotoxigenic E. coli, respectively. While 14 (82.35%) DEC isolates harbored the stx2 gene, five (29.41%) the eae gene, five (29.41%) the hlyA gene and three (17.65%) harbored the st gene. All the DEC isolates were resistant to erythromycin and vancomycin; whereas, they were susceptible to ampicillin, nalidixic acid and norfloxacin. Furthermore, 64.7% of DEC isolates showed resistance to both ceftazidime and kanamycin and 88.24% of the isolates showed multidrug resistance.ConclusionThis study detected DEC isolates having different virulence genes, which showed single and multiple antimicrobial resistance. Given the existing poor hygienic and sanitary practices along the abattoir-to-table food chain, coupled with the habit of raw meat consumption, this result indicates a potential public and animal health risk from the pathogen and antimicrobial resistance.

Molecular Characterization and Antimicrobial Profiling of Escherichia coli isolates from Indian Camelus

Background: Escherichia coli is frequently associated with multiple antimicrobial resistances and a major cause of bacterial extraintestinal infections in livestock and humans. Escherichia coli resides in the lower digestive tract as harmless commensals but a subset of E. coli strains has acquired the ability due to acquisition of virulence and antibiotic genes, cause intestinal or extraintestinal diseases. Methods: In this field-laboratory investigation during 2020-2021, samples were taken from different localities of Bikaner district and surrounding are of Rajasthan. A total no. of 70 fecal samples were collected and immediately transferred to the Department of Veterinary Microbiology. In the laboratory, the collected samples were further processed for isolation and identification of E. coli bacteria. Result: Confirmation of E. coli was done using primary and biochemical test which are screened for hemolysin property, biofilm formation, antibiogram study and antibiotic resistance gene. All the isolates of E. coli used to show characteristic metallic sheen on EMB agar plate and excellent identification was done with VITEK 2 system. All the camel isolates shown partial hemolysis on sheep blood agarand 45.71% camel isolates were positive for biofilm formation. All these 70 E. coli isolates from camel were resistant to Penicillin (94.38%) which was followed by amoxicillin+sulbactam (85.71%), erythromycin (71.14%), cefixime+clavulanic acid (71.43%). Highest sensitivity to chloramphenicol (81.28%) followed by sulphadiazine (48.57%) and cotrimoxazole (48.28%). All these 70 isolates were screened for antibiotic resistance genes. On the basis of molecular screening of the antibiotic resistance genes, majority of the isolates carried BlaTEM gene in camel (56/70; 80%), followed by StrA (29/70; 41.40%), Sul-3 in (22/70; 31.42%), Sul-2 (18/70; 25.71%), aadA (28/70; 40%), tet(B) (22/70; 31.42%) isolates.

Global transmission of extended-spectrum cephalosporin resistance in Escherichia coli driven by epidemic plasmids

Extended-spectrum cephalosporins (ESCs) are third and fourth generation cephalosporin antimicrobials used in humans and animals to treat infections due to multidrug-resistant (MDR) bacteria. Resistance to ESCs (ESC-R) in Enterobacterales is predominantly due to the production of extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamases (AmpCs). The dynamics of ESBLs and AmpCs are changing across countries and host species, the result of global transmission of ESC-R genes. Plasmids are known to play a key role in this dissemination, but the relative importance of different types of plasmids is not fully understood. In this study, Escherichia coli with the major ESC-R genes blaCTX-M-1, blaCTX-M-15, blaCTX-M-14 (ESBLs) and blaCMY-2 (AmpC), were selected from diverse host species and other sources across Canada, France and Germany, collected between 2003 and 2017. To examine in detail the vehicles of transmission of the ESC-R genes, long- and short-read sequences were generated to obtain complete contiguous chromosome and plasmid sequences (n=192 ESC-R E.coli). The types, gene composition and genetic relatedness of these plasmids were investigated, along with association with isolate year, source and geographical origin, and put in context with publicly available plasmid sequences. We identified five epidemic resistance plasmid subtypes with distinct genetic properties that are associated with the global dissemination of ESC-R genes across multiple E.coli lineages and host species. The IncI1 pST3 blaCTX-M-1 plasmid subtype was found in more diverse sources than the other main plasmid subtypes, whereas IncI1 pST12 blaCMY-2 was more frequent in Canadian and German human and chicken isolates. Clonal expansion also contributed to the dissemination of the IncI1 pST12 blaCMY-2 plasmid in ST131 and ST117 E.coli harbouring this plasmid. The IncI1 pST2 blaCMY-2 subtype was predominant in isolates from humans in France, while the IncF F31:A4:B1 blaCTX-M-15 and F2:A-:B- blaCTX-M-14 plasmid subtypes were frequent in human and cattle isolates across multiple countries. Beyond their epidemic nature with respect to ESC-R genes, in our collection almost all IncI1 pST3 blaCTX-M-1 and IncF F31:A4:B1 blaCTX-M-15 epidemic plasmids also carried multiple antimicrobial resistance (AMR) genes conferring resistance to other antimicrobial classes. Finally, we found genetic signatures in the regions surrounding specific ESC-R genes, identifying the predominant mechanisms of ESC-R gene movement, and using publicly available databases, we identified these epidemic plasmids from widespread bacterial species, host species, countries and continents. We provide evidence that epidemic resistance plasmid subtypes contribute to the global dissemination of ESC-R genes, and in addition, some of these epidemic plasmids confer resistance to multiple other antimicrobial classes. The success of these plasmids suggests that they may have a fitness advantage over other plasmid types and subtypes. Identification and understanding of the vehicles of AMR transmission are crucial to develop and target strategies and interventions to reduce the spread of AMR. This project was supported by the Joint Programming Initiative on Antimicrobial Resistance (JPIAMR), through the Medical Research Council (MRC, MR/R000948/1), the Canadian Institutes of Health Research (CFC-150770), and the Genomics Research and Development Initiative (Government of Canada), the German Federal Ministry of Education and Research (BMBF) grant no. 01KI1709, the French Agency for food environmental and occupational health & safety (Anses), and the French National Reference Center (CNR) for antimicrobial resistance. Support was also provided by the Biotechnology and Biological Sciences Research Council (BBSRC) through the BBSRC Institute Strategic Programme Microbes in the Food ChainBB/R012504/1 and its constituent project BBS/E/F/000PR10348 (Theme 1, Epidemiology and Evolution of Pathogens in the Food Chain).

Comparative analysis of antimicrobial resistance phenotype and genotype of Riemerella anatipestifer

Riemerella anatipestifer is one of the important bacterial pathogens that threaten the waterfowl farming industry. In this study, 157 suspected R. anatipestifer strains were isolated from diseased ducks and geese from seven regions of China during 2019–2020, and identified using multiple polymerase chain reaction (PCR). Antimicrobial susceptibility tests and whole-genome sequence (WGS) analysis were then performed for comparative analysis of antimicrobial resistance phenotypes and genotypes. The results showed that these strains were susceptible to florfenicol, ceftriaxone, spectinomycin, sulfafurazole and cefepime, but resistant to kanamycin, amikacin, gentamicin, and streptomycin, exhibiting multiple antimicrobial resistance phenotypes. WGS analysis revealed a wide distribution of genotypes among the 157 strains with no apparent regional pattern. Through next-generation sequencing analysis of antimicrobial resistance genes, a total of 88 resistance genes were identified. Of them, 19 tetracycline resistance genes were most commonly found, followed by 15 efflux pump resistance genes, 11 glycopeptide resistance genes and seven macrolide resistance genes. The 157 R. anatipestifer strains contained 42–55 resistance genes each, with the strains carrying 47 different resistance genes being the most abundant. By comparing the antimicrobial resistance phenotype and genotype, it was observed that a high correlation between them for most antimicrobial resistance properties was detected, except for a difference in aminoglycoside resistance phenotype and genotype. In conclusion, 157 R. anatipestifer strains exhibited severe multiple antimicrobial resistance phenotypes and genotypes, emphasizing the need for improved antimicrobial usage guidelines. The wide distribution and diverse types of resistance genes among these strains provide a foundation for studying novel mechanisms of antimicrobial resistance.

In-depth characterization of multidrug-resistant NDM-1 and KPC-3 co-producing Klebsiella pneumoniae bloodstream isolates from Italian hospital patients.

Bloodstream infection (BSI) caused by carbapenem-resistant Klebsiella pneumoniae (KP) poses significant challenges, particularly when the infecting isolate carries multiple antimicrobial resistance (AMR) genes/determinants. This study, employing short- and long-read whole-genome sequencing, characterizes six New Delhi metallo-β-lactamase (NDM) 1 and KP carbapenemase (KPC) 3 co-producing KP isolates, the largest cohort investigated in Europe to date. Five [sequence type (ST) 512] and one (ST11) isolates were recovered from patients who developed BSI from February to August 2022 or February 2023 at two different hospitals in Rome, Italy. Phylogenetic analysis revealed two distinct clusters among ST512 isolates and a separate cluster for the ST11 isolate. Beyond blaNDM-1 and blaKPC-3, various AMR genes, indicative of a multidrug resistance phenotype, including colistin resistance, were found. Each cluster-representative ST512 isolate harbored a blaNDM-1 plasmid (IncC) and a blaKPC-3 plasmid [IncFIB(pQil)/IncFII(K)], while the ST11 isolate harbored a blaNDM-1 plasmid [IncFII(pKPX1)] and a blaKPC-3 plasmid [IncFIB(K)/IncFII(K)]. The blaNDM-1 plasmids carried genes conferring resistance to clinically relevant antimicrobial agents, and the aminoglycoside resistance gene aac(6')-Ib was found on different plasmids. Colistin resistance-associated mgrB/pmrB gene mutations were present in all isolates, and the yersiniabactin-encoding ybt gene was unique to the ST11 isolate. In conclusion, our findings provide insights into the genomic context of blaNDM-1/blaKPC-3 carbapenemase-producing KP isolates.IMPORTANCEThis study underscores the critical role of genomic surveillance as a proactive measure to restrict the spread of carbapenemase-producing KP isolates, especially when key antimicrobial resistance genes, such as blaNDM-1/blaKPC-3, are plasmid borne. In-depth characterization of these isolates may help identify plasmid similarities contributing to their intra-hospital/inter-hospital adaptation and transmission. Despite the lack of data on patient movements, it is possible that carbapenem-resistant isolates were selected to co-produce KP carbapenemase and New Delhi metallo-β-lactamase via plasmid acquisition. Studies employing long-read whole-genome sequencing should be encouraged to address the emergence of KP clones with converging phenotypes of virulence and resistance to last-resort antimicrobial agents.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum β-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

Production and immunological characterisation of recombinant flagellin C of <i>Pseudomonas aeruginosa</i>

SCIENTIFIC RELEVANCE. Pseudomonas aeruginosa is one of the main causative agents of focal and diffuse suppurative inflammations in immunocompromised individuals. The multiple antimicrobial resistance of Р. aeruginosa has created an urgent need to develop effective preventive vaccines against this bacterium and to enhance their protective properties by selecting adjuvants. A promising strategy is to use flagellin, a P. aeruginosa flagellum component inducing the innate immune system through interaction with toll-like receptor 5 (TLR5) and activating the T-cell immune response, as a component or adjuvant in vaccine development.AIM. This study aimed to produce recombinant flagellin C (FliC) of P. aeruginosa and investigate its immunogenicity, adjuvanticity, and protective properties.MATERIALS AND METHODS. The fliC nucleotide sequence was obtained by PCR on template DNA of P. aeruginosa PA-103 and was inserted into the pQE-30 plasmid for subsequent expression in Escherichia coli M15. Recombinant FliC purification involved two stages: isolation of inclusion bodies and their dissolution in buffers containing urea and guanidine hydrochloride. Mice were immunised by two intraperitoneal injections with a two-week interval. The immunisation used purified recombinant FliC at a dose of 50 μg per animal and its combination with the Klebsiella pneumoniae surface antigen in a 1:1 ratio. Serum samples from immunised mice were tested for specific antibodies to recombinant FliC by enzyme-linked immunosorbent assay (ELISA). The protective properties of FliC were assessed by intraperitoneal challenge of mice with cultures of P. aeruginosa PA-103 and K. pneumoniae 204.RESULTS. The authors obtained the producing strain, generated recombinant FliC, and purified the protein to a 97.6% purity. The analysis of serum samples from immunised mice by ELISA and the protection assessment in challenge experiments showed that the purified recombinant FliC protein had immunogenic properties. Furthermore, the experimental challenge of FliC-immunised mice with P. aeruginosa confirmed that recombinant FliC had protective properties, as evidenced by the protection index of 3.0. Recombinant FliC exhibited adjuvant properties, as demonstrated by the effectiveness index of 6.1 observed in the experimental challenge of mice immunised with the combination of recombinant FliC and the surface antigen of K. pneumoniae.CONCLUSIONS. The purified recombinant FliC protein of P. aeruginosa demonstrated protective activity in mice challenged with P. aeruginosa and adjuvant properties when combined with the K. pneumoniae surface antigen, increasing the immunogenicity of the latter. The use of recombinant FliC holds promise for the creation of a candidate vaccine against P. aeruginosa infection.

Complete genome sequences of a Canadian strain of enteroaggregative Escherichia coli (EAEC) with multiple metals and antimicrobial resistance genes isolated from municipal waste-activated sludge.

Enteroaggregative Escherichia coli (EAEC) is an emerging food-borne pathogen causing acute or persistent diarrhea in humans. Here, we report the complete genome sequence of a strain of EAEC with multiple metals and antimicrobial resistance genes isolated from a waste-activated sludge collected from a Canadian municipal wastewater treatment plant.

An investigation of the presence and antimicrobial susceptibility of Enterobacteriaceae in raw and cooked kibble diets for dogs in the United Kingdom.

Raw meat diets (RMD) for dogs are an increasingly popular alternative pet food choice, however studies worldwide have demonstrated them to be contaminated with zoonotic and antimicrobial resistant (AMR) bacteria, including bacteria resistant to critically important antibiotics. Despite this, few data exist surrounding the presence of these bacteria in RMD in the United Kingdom. The present study aimed to identify the most commonly selected RMD and non-raw diets (NRMD) by United Kingdom dog owners. Additionally, it investigated the presence of AMR-Enterobacteriaceae in samples of pre-prepared RMD and cooked commercial kibble dog foods. An online survey investigating diet preferences of United Kingdom dog owners was open for 6 weeks between February-March 2020. From this, the top 10 brands of pre-prepared raw and cooked kibble diets were ascertained and 134 samples purchased (110 RMD, 24 kibble) and subjected to microbiological testing. Bacterial enumeration of E. coli and other Enterobacteriaceae was undertaken, and the presence of Salmonella spp. and AMR-E. coli within samples determined. Whole genome sequencing was undertaken on Salmonella spp. and third-generation cephalosporin-resistant 3GCR-E. coli isolates. Pre-prepared RMD was most commonly selected by dog owners who fed RMD, and cooked commercial complete dry food was most frequently fed by owners who fed NRMD. Damaged and leaking packaging was observed in samples of RMD, alongside variability in information provided surrounding product traceability. Counts of E. coli and other Enterobacteriaceae exceeding >5,000 CFU/g were identified in samples of RMD. AMR-, extended-spectrum beta-lactamase (ESBL)-producing and 3GCR-E. coli was isolated from 39, 14 and 16% of RMD samples, respectively. Multiple antimicrobial resistance genes were identified in 3GCR-E. coli isolates. Of the ESBL encoding genes, blaCTX-M-15 was most commonly identified. S. enterica was isolated from 5% of RMD samples. No Enterobacteriaceae were isolated from any of the cooked kibble samples. The present study suggests that pre-prepared RMD available for dogs in the United Kingdom can be contaminated with zoonotic and AMR-Enterobacteriaceae. RMDs, therefore, are potentially an important One Health concern. Veterinary and medical professionals, pet food retailers and pet owners should be aware of these risks; and stringent hygiene measures should be practiced if owners choose to feed RMD.

Emergence and genomic characteristics of multi-drug-resistant Salmonella in pet turtles and children with diarrhoea.

Pet turtles are a well-recognized source of human salmonellosis, posing a threat to human health, particularly children who commonly keep pet turtles. To date, the genomic characteristics of Salmonella among pet turtles and children has not been well described. We investigated the prevalence, antimicrobial resistance (AMR) and genomic characteristics of Salmonella from pet turtles in Beijing, China. In total, 9.6 % (46/480) of pet turtles were positive for Salmonella with S. Thompson being the dominant serovar (19/46) in 2019. Moreover, 80.4 % of Salmonella were multi-drug resistant (MDR) and 60.7 % were resistant to ampicillin, streptomycin, sulfonamides and tetracycline (ASSuT). We further compared the genomes of S. Thompson isolates from pet turtles (n=19) with those from children with diarrhoea (n=28) in the same region and year, most of which were sequence type (ST)26, with one novel ST7937 identified from a child-associated isolate. S. Thompson isolates from children with diarrhoea exhibited less resistance than isolates from pet turtles. Most MDR isolates possessed multiple AMR genes, including the AmpC β-lactamase-encoding genes bla DHA-15 and bla DHA-1 which co-occurred with the IncA/C and IncHI plasmid replicon types. To the best of our knowledge, this is the first time that the bla DHA-15 gene has been detected from Salmonella. Several pet turtle-associated S. Thompson isolates comprised phylogenetically close clusters with those from children with diarrhoea (<20 SNP differences). Bayesian analysis demonstrated that the Chinese ST26 S. Thompson strains had a recent evolutionary history and evolved into two major clades, with one clade acquiring various resistant plasmids. Our findings revealed the emergence of MDR Salmonella among pet turtles in China and provided evidence for the interspecies transmission of S. Thompson.

The Association between Transformation Ability and Antimicrobial Resistant Potential in Haemophilus influenzae.

The prevalence of quinolone low-susceptible Haemophilus influenzae has increased in Japan. Low quinolone susceptibility is caused by point mutations in target genes; however, it can also be caused by horizontal gene transfer via natural transformation. In this study, we examined whether this horizontal gene transfer could be associated with resistance to not only quinolones but also other antimicrobial agents. Horizontal transfer ability was quantified using the experimental transfer assay method for low quinolone susceptibility. Further, the association between horizontal transfer ability and resistance to β-lactams, the first-choice drugs for H. influenzae infection, was investigated. The transformation efficiency of 50 clinical isolates varied widely, ranging from 102 to 106 colony forming unit (CFU) of the colonies obtained by horizontal transfer assay. Efficiency was associated with β-lactam resistance caused by ftsI mutations, indicating that strains with high horizontal transfer ability acquired quinolone low-susceptibility as well as β-lactam resistance more easily. Strains with high transformation efficiency increased the transcript level of comA, suggesting that enhanced com operon was associated with a high DNA uptake ability. Overall, this study revealed that the transformation ability of H. influenzae was associated with multiple antimicrobial resistance. Increase in the number of strains with high horizontal transformation ability has raised concerns regarding the rapid spread of antimicrobial-resistant H. influenzae.

Transcriptional responses of cytokines, immunoglobulin A, and nitric oxide genes in 1-day-old chicks post Salmonella typhimurium infection: An experimental study.

Salmonella has become one of the hazards prevalent foodborne pathogens causing different diseases in chickens. However, Salmonella typhimurium (ST), a nonhost-specific serovar, is a major avian agent that causes severe disturbance in young chicken wellness. The occurrence of Salmonella in chickens and their antimicrobial resistance were explored in this study. In addition, the immune response of 1-day-old broiler chicks, against multidrug resistant (MDR) ST infection, was also assessed at 4 and 24 hours post infection (pi) in the cecum and spleen, representing their mucosal and systemic immune responses, respectively. A total of 375 samples from 130 diseased and apparently healthy broiler and layer chickens were randomly collected for Salmonella isolation, identification, and resistance profile evaluation, from farms and different clinical laboratories. The immune response of 1-day-old broiler chicks, Ross 308, against in-vivo ST infection was ascertained through the evaluation of heterophile phagocytosis and s expression of cytokines, immunoglobulin A and other immune-regulating genes in the cecum and spleen. Twenty-four, 1-day-old nonvaccinated broiler chicks were used and divided into two groups. The chicks in the infected group were orally inoculated with 0.5 ml of 2 × 108 colony forming units (CFU)/ml of MDR ST suspension, while those in the control group were taken nutrient broth. Seven out of 130 (5.38%) examined chickens were positive for Salmonella. All isolates (100%) were resistant to amoxicillin-clavulanic acid (AMC), cefazolin (CZ), cefoxitin (FOX), ciprofloxacin (CIP), nalidixic acid (NA), tetracycline (TE), fosfomycin (FOS), and colistin (CT) with multiple antimicrobial resistances (MARs) index range of 0.72-0.83, where none of them was resistant to meropenem (MEM). The results of immune response revealed that chicks infected with ST showed significantly different phagocytosis percentages and index values compared to controls. According to the real-time quantitative polymerase chain reaction (RT-qPCR) results, the transcription of IL-8, iNOS, IL-18, IgA, and IFN-γ for chicks infected by ST showed a significantly increased trend (p < 0.01) with increasing chicken age and was higher in the cecum than spleen compared to controls (p < 0.05) during 24 hours after infection. The findings indicated a strong mucosal immune response in the chicks after the ST challenge, which reflects humoral and cellular responses. Our insight recommended the occurrence of a natural immune response stimulator at 1 day age to face the infection, and this can prevent the resistance transfer, with efficient control measures.

Sequencing and multiple antimicrobial resistance of Pseudomonas fluorescens isolated from Nile tilapia fish in Egypt.

Pseudomonas fluorescens is one of the main causes of septicemic diseases among freshwater fish, causing severe economic losses and decreasing farm efficiency. Thus, this research was aimed to investigate the occurrence of P. fluorescens in Nile Tilapia (O. niloticus) fish in Egypt, gene sequencing of 16SrDNA gene, and antimicrobial susceptibility. P. fluorescens strains were detected in 32% (128\400) of apparently healthy (9%; 36\400) and diseased (23%; 92\400) Nile tilapia fish. The highest prevalence was observed in gills of fish, 31.3% followed by intestine 26.9%, liver 24.2%, and kidneys 17.6%. The PCR results for the 16SrDNA gene of P. fluorescens showed 16SrDNA gene in 30% of examined isolates. Moreover, Homogeny and a strong relationship between strains of P. fluorescens was confirmed using 16SrDNA sequences. Beside the responsibility of 16SrDNA gene on the virulence of P. fluorescens. The results of antimicrobial susceptibility tests revealed that all strains were resistant to piperacillin (100%), followed by ceftazidime (29.7%), and cefepime (25.8%). The strains of P. fluorescence were highly sensitive to cefotaxime (74.2%), followed by ceftriaxone and levofloxacin (70.3% each). Interestingly, 29.7% of strains of P. fluorescens were multiple antimicrobial-resistant (MAR).

A TetR family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae.

Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a resistance-nodulation-cell division (RND) efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB, or tolC abrogated the ARS resistance of the atrR mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a resistance-nodulation-cell division (RND) efflux pump of V. cholerae, is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.

Oxidative stress, gene expression and histopathology of cultured gilthead sea bream (Sparus aurata) naturally co-infected with Ergasilus sieboldi and Vibrio alginolyticus

BackgroundParasitic and bacterial co-infections have been associated with increasing fish mortalities and severe economic losses in aquaculture through the past three decades. The aim of this study was to evaluate the oxidative stress, histopathology, and immune gene expression profile of gilthead sea bream (Sparus aurata) co-infected with Ergasilus sieboldi and Vibrio alginolyticus.ResultsVibrio alginolyticus and Ergasilus sieboldi were identified using 16 S rRNA and 28 S rRNA sequencing, respectively. The collagenase virulence gene was found in all Vibrio alginolyticus isolates, and the multiple antimicrobial resistance index ranged from 0.286 to 0.857. Oxidant-antioxidant parameters in the gills, skin, and muscles of naturally infected fish revealed increased lipid peroxidation levels and a decrease in catalase and glutathione antioxidant activities. Moreover, naturally co-infected gilthead sea bream exhibited substantial up-regulation of il-1β, tnf-α, and cyp1a1. Ergasilus sieboldi encircled gill lamellae with its second antennae, exhibited severe gill architectural deformation with extensive eosinophilic granular cell infiltration. Vibrio alginolyticus infection caused skin and muscle necrosis in gilthead sea bream.ConclusionThis study described some details about the gill, skin and muscle tissue defense mechanisms of gilthead sea bream against Ergasilus sieboldi and Vibrio alginolyticus co-infections. The prevalence of co-infections was 100%, and no resistant fish were detected. These co-infections imbalance the health status of the fish by hampering the oxidant-antioxidant mechanisms and proinflammatory/inflammatory immune genes to a more detrimental side. Our results suggest that simultaneous screening for bacterial and parasitic pathogens should be considered.

Prospects for the use of probiotic organisms to develop alternative strategies for disinfection and prevention of infectious diseases

Introduction: An important feature of microorganisms is the ability to adapt to adverse external influences, including the development of multiple antimicrobial resistance, leading to such negative consequences for humans as nosocomial infections, problems with cleaning rooms, medical and industrial equipment. The active use of chemical disinfectants for high-quality disinfection and cleaning has a number of disadvantages, including the risks of pathogens with new mechanisms of resistance to antimicrobial agents. The search for new, effective and safe antimicrobial agents as an alternative to chemical disinfectants is relevant. The use of probiotic strains of microorganisms, including lactic acid bacteria, in this capacity may be a promising area of research.Objective: to analyze the literature data on studies of the potential of probiotic organisms, including lactic acid bacteria, to develop alternative disinfection and prevention strategies.Materials and methods: This review of the subject field has been prepared based on the guiding principles of PRISMA-ScR. SCOPUS, Google Scholar, and RSCI databases were used. The works in Russian and English were considered, for the period 1995-2023.Results: The review included 89 articles exploring the disadvantages of traditional disinfection methods and the search for alternative sanitation strategies. The description of various solutions for the introduction of probiotic purification methods is systematized – using bacteriophages, probiotic microorganisms of R. Bacillus and representatives of lactic acid bacteria as active agents.Conclusions: A system of sanitary measures based on probiotic biodesinfectants, including lactic acid bacteria, can be included among the tools for countering pathogens, including their biofilms and forms with multidrug resistance. The introduction of a probiotic hygienic cleaning system, without having a negative impact on the environment, can increase the effectiveness of traditional hygienic preventive measures both in medical institutions and in production.

Rapid Detection of Antimicrobial Resistance Genes in Critically Ill Children Using a Custom TaqMan Array Card.

Bacteria are identified in only 22% of critically ill children with respiratory infections treated with antimicrobial therapy. Once an organism is isolated, antimicrobial susceptibility results (phenotypic testing) can take another day. A rapid diagnostic test identifying antimicrobial resistance (AMR) genes could help clinicians make earlier, informed antimicrobial decisions. Here we aimed to validate a custom AMR gene TaqMan Array Card (AMR-TAC) for the first time and assess its feasibility as a screening tool in critically ill children. An AMR-TAC was developed using a combination of commercial and bespoke targets capable of detecting 23 AMR genes. This was validated using isolates with known phenotypic resistance. The card was then tested on lower respiratory tract and faecal samples obtained from mechanically ventilated children in a single-centre observational study of respiratory infection. There were 82 children with samples available, with a median age of 1.2 years. Major comorbidity was present in 29 (35%) children. A bacterial respiratory pathogen was identified in 13/82 (16%) of children, of which 4/13 (31%) had phenotypic AMR. One AMR gene was detected in 49/82 (60%), and multiple AMR genes were detected in 14/82 (17%) children. Most AMR gene detections were not associated with the identification of phenotypic AMR. AMR genes are commonly detected in samples collected from mechanically ventilated children with suspected respiratory infections. AMR-TAC may have a role as an adjunct test in selected children in whom there is a high suspicion of antimicrobial treatment failure.

Microbiological assessment and Multiple Antimicrobial Resistance Profile of Enterobacteriaceae recovered from kulikuli sold at selected markets in Ife-North Local Government Area

Microbiological assessment and Multiple Antimicrobial Resistance Profile of Enterobacteriaceae recovered from kulikuli sold at selected markets in Ife-North Local Government Area

Whole genome sequence and comparative genomics analysis of multidrug-resistant Staphylococcus xylosus NM36 isolated from a cow with mastitis in Basrah city

BackgroundStaphylococcus xylosus is a coagulase-negative, gram-positive coccus that is found in the environment and as a commensal organism on the skin and mucosal surfaces of animals. Despite the fact that S. xylosus is considered a nonpathogenic bacterium, several studies have linked S. xylosus to opportunistic infections in both animals and humans. During an investigation of mastitis-causing agents in the governorate of Basrah, Iraq, we identified an antibiotic-resistant strain of S. xylosus NM36 from a milk sample from a cow with chronic mastitis. In addition to robust biofilm formation, multiple antibiotic resistance phenotypes were found. To further understand the genetic background for these phenotypes, the full genome of S. xylosus NM36 was analyzed. ResultsThe genome consisted of a single circular 2,668,086 base pairs chromosome containing 32.8% G + C. There were 2454 protein-coding sequences, 4 ribosomal RNA (rRNA) genes, and 50 transfer RNA (tRNA) genes in the genome. In addition, genetic variation was studied by searching sequence data against a representative reference genome. Consequently, single-nucleotide polymorphism analysis was conducted and showed that there were 46,610 single-nucleotide polymorphisms (SNPs), 523 insertions, and 551 deletions. In order to overcome antibiotics, S. xylosus NM36 had been armed with several antibiotic resistance genes from several groups and families. The genome annotation service in PathoSystems Resource Integration Center (PATRIC) and Rapid Annotation using Subsystem Technology (RAST) annotation servers showed that there are multiple antimicrobial resistance elements, including antibiotic inactivation enzymes (BlaZ family, FosB), antibiotic resistance gene clusters (TcaB, TcaB2, TcaR), proteins involved in methicillin resistance (LytH, FmtA, FemC, HmrB, HmrA), TetR family transcriptional regulators, and efflux pumps conferring antibiotic resistance (NorA). In addition, we investigated and categorized the biofilm and quorum-sensing elements of the NM36 strain and found that it has multiple subsets of biofilm regulators, confirming its pathogenic nature. ConclusionsThese findings necessitate a reevaluation of microbial and clinical interventions when dealing with coagulase-negative staphylococci, particularly in the context of studies pertaining to public health. This is the first time, to our knowledge, that the entire genome of S. xylosus has been sequenced in Iraq.

MOLECULAR IDENTIFICATION AND ANTIBIOGRAM OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS FROM WOUNDS OF BOTH IN- AND OUT-PATIENTS AT UNIVERSITY OF BENIN TEACHING HOSPITAL (UBTH), BENIN CITY, NIGERIA

Methicillin-resistant Staphylococcus aureus (MRSA) stands as a prominent bacterial pathogen associated with various human infections, characterized by its remarkable ability to rapidly develop resistance to multiple antibiotics. The study aimed to characterize MRSA isolated from wounds among hospital patients. A total of eighty-seven unique S. aureus isolates were obtained from wound cultures collected from patients at the University of Benin Teaching Hospital (UBTH) in Benin City, Nigeria. These samples underwent assessment for methicillin resistance through conventional cultural methods, and detection of methicillin resistance (mecA) and S. aureus specific (nuc) genes using PCR amplification. The MRSA isolates underwent assessment for antimicrobial resistance through disc diffusion testing and for virulence factors using phenotypic testing procedures. An overall prevalence of 34/87 (39.1%) MRSA isolates was recorded. Based on the source of the isolate, the distribution of MRSA isolates from in-patients was 22/56 (39.3%) and out-patients was 12/31 (39.7%). Total Panton valentine leucocidin (PVL) screening revealed 11/34 (32.4%). All the isolates were resistant to penicillin 34/34 (100%) and cefepime 34/34 (100%). Lower level of resistance was demonstrated towards ciprofloxacin 3/34 (8.8%). Overall, every isolate displayed resistance to at least three antibiotics, resulting in a multiple antimicrobial resistance index of at least 0.33. The total virulence factors observed were beta-hemolytic activity 22/34 (64.7%), protease activity 14/34 (41.2%), and lipase activity 19/34 (55.9%). This study emphasized the significance of regularly monitoring MRSA prevalence within both the hospital and the local community. Therefore, the need for better intervention and continuous enlightenment on risks associated with the abuse...