fosA3 Genes Research Articles

Characterization of antibiotic resistance genes and mobile elements in extended-spectrum β-lactamase-producing Escherichia coli strains isolated from hospitalized patients in Guangdong, China.

This study aimed to investigate the high-resolution phenotypic and genotypic characterization of extended-spectrum β-lactamase (ESBL)-producing E. coli strains isolated from hospitalized patients to explore the resistance genes and mobile genetic elements (MGEs) involved in horizontal dissemination. Between May and September 2021, a total of 216 ESBL-producing E. coli isolates were recovered from multiple departments. The identification of strains was performed using MALDI-TOF mass spectrometry and PCR, while antibiotic susceptibility testing was carried out using the Vitek 2 COMPACT system to determine resistance patterns, while PCR was used to detect different resistance genes and MGEs. In addition, a conjugation assay was performed to investigate the horizontal gene transfer of resistance genes. Selected isolates underwent whole-genome sequencing using the Illumina MiSeq platform. A total of 216 out of 409 E. coli isolates recovered from a tertiary hospital were observed to be ESBL-producing, giving a carriage rate of 52.8%, as determined by phenotypic screening. The most frequent sources of ESBL-producing E. coli isolates were urine (129/216, 59.72%,) and blood (50/216, 23.14%). The most prevalent ESBL genes identified were blaCTX-M (60.18%), blaTEM (40.27%), and blaSHV (18.05%). Three E. coli isolates were found to carry the genes blaNDM, mcr-1, and fosA3 genes. The most prevalent MGEs were IS26 (95.37%), Int (87.03%), and IncFIB (76.85%). Whole-genome sequencing analysis of eight MDR E. coli strains revealed that these isolates belonged to eight different sequence types (STs) and serotypes and were found to harbor multiple plasmid replicons and virulence factors. This study highlights a high incidence of antibiotic resistance genes and MGEs associated with the dissemination of ESBLs and other resistance genes.

Highly drug resistant clone of Salmonella Kentucky ST198 in clinical infections and poultry in Zimbabwe

A highly multidrug-resistant strain of Salmonella enterica serotype Kentucky (S. Kentucky) of sequence type (ST)198 emerged in North Africa and has since spread widely. To investigate the genetic diversity and phylogenetic relationship of S. Kentucky in Zimbabwe and identify potential sources of infection, the whole-genome sequence of 37 S. Kentucky strains isolated from human clinical infections and from poultry farms between 2017 and 2020 was determined. Of 37 S. Kentucky isolates, 36 were ST198 and one was ST152. All ST198 isolates had between six and fifteen antimicrobial resistance (AMR) genes, and 92% carried at least ten AMRs. All ST198 isolates harbored the Salmonella genomic island K-Israel variant (SGI1-KIV) integrated into the chromosome with aac(3)-ld, aac(6)-laa, aadA7, blaTEM-1, sul1, and tetA genes, with occasional sporadic loss of one or more genes noted from five isolates. All ST198 isolates also had mutations in the quinolone resistance-determining region of the gyrA and parC genes. The blaCTX-M-14.1 and fosA3 genes were present in 92% of the ST198 isolates, conferring resistance to extended-spectrum cephalosporins and fosfomycin, respectively, were present on an IncHI2 plasmid with the aadA2b, aadA1, aph(3’)-Ib, aph(6’)-Id, cmlA1 and sul3 AMR genes. S. Kentucky ST198 isolates from Zimbabwe formed a closely related phylogenetic clade that emerged from a previously reported global epidemic population. The close genetic relationship and population structure of the human clinical and poultry isolates of ST198 in Zimbabwe are consistent with poultry being an important source of highly resistant strains of S. Kentucky in Zimbabwe.

Emergence of fosfomycin resistance among isolates of multidrug-resistant Enterobacter species at a tertiary care hospital in Dhaka, Bangladesh

Background: The emergence of multidrug-resistant Enterobacter species as a worrying resistant pathogen seriously threatened human health. The rising rate of resistance to commonly used antibiotics limit the choice hence it is urgent to evaluate the antimicrobial activity of older drug like Fosfomycin. Objective: The study aimed to seek the frequency of fosfomycin resistance in the clinical isolates of Enterobacter species and to detect the fosfomycin resistance gene along with antibiotic resistance pattern. Methods: This cross-sectional study was conducted in the Department of Microbiology of a tertiary care hospital in Bangladesh from July 2018 to June 2019. Enterobacter spp. was isolated from a total of 350 samples by a standard microbiological method. Antibiotic susceptibility was performed by the disk diffusion technique. Antibiotic susceptibility and minimum inhibitory concentration (MIC) of fosfomycin were determined by the agar dilution method. Fosfomycin resistance gene fosA, fosA3, fosA4, fosA5, fosB, fosB2, fosC, fosC2 and fosX among fosfomycin‑resistant Enterobacter spp detected by polymerase chain reaction (PCR) using specific primer. Sequencing of fosA and fosA₅ was performed by capillary method, and the nucleotide sequence of fosA₅ has been deposited to GenBank. Results: Out of 28 Enterobacter spp. 7 (25%) fosfomycin resistant Enterobacter spp. were detected by agar dilution method. Out of 7 fosfomycin-resistant strains, 4 (57.14%) were isolated from urine samples. Fifteen (53.57%) isolates of Enterobacter spp. were multidrug-resistant detected by disc diffusion technique. All of the fosfomycin-resistant isolates were MDR. A significant rise in the MIC was found between 256µg/ml- ≥4096 µg/ml to fosfomycin. PCR revealed that 100% of fosfomycin resistant isolates are positive for fosA, 71.43% and 28.57% were positive for fosA₅ and fosB₂ respectively. Sequencing of fosA₅ gene established the FosA family fosfomycin resistance glutathione transferase gene. Conclusion: The results of this study showed a high proportion of fosfomycin resistance among multidrug-resistant Enterobacter spp. irrespective of fosfomycin usage in Bangladesh. FosA family fosfomycin resistance glutathione transferase gene emerging in Bangladesh. Bangladesh Medical Res Counc Bull 2022; 48(3): 203-210

Polyclonal Spread of Fosfomycin Resistance among Carbapenemase-Producing Members of the Enterobacterales in the Czech Republic.

Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.

Fosfomycin Resistant Enterobacterales IsolatedFrom Chicken Meat in Turkey

This study was conducted to obtain the relative prevalence of fosfomycin resistant (FOSr) Enterobacterales in raw chicken meat samples in Turkey. Samples (n=85) were enriched in non-selective media and transferred to MacConkey agar plates containing FOS and glucose-6-phosphate. As a result, FOSr Enterobacterales isolates were detected by a selective method in 27% of raw chicken meat samples (n=23) and identified as Escherichia coli (21/26), Klebsiella oxytoca (2/26), Escherichia vulneris (1/26), Raoultella terrigena (1/26) and Kluyvera intermedia (1/26). PFGE analysis showed 16 different band patterns in Escherichia spp. isolates (n=22) based on the 85% similarity. The minimum inhibitory concentration for FOS against all isolates was determined to be ≥64 mg/L. In addition, the highest rate of resistance was determined for nalidixic acid (72.7%), ampicillin (68.2%), tetracycline (59.1%), trimethoprim-sulfamethoxazole (54.5%), and chloramphenicol (59.1%) among all Escherichia isolates. PCR screening and sequencing identified the presence of fosA4 and fosA3 genes in ten (47.6%) and seven (33.3%) E. coli isolates, respectively. The fosA3 gene has also appeared in K. intermedia, and R. terrigena isolates. Only two E. coli isolates were positive for the blaCTX-M-55 gene, whereas the aac(6')-Ib-cr gene was identified in eight E. coli and one K. intermedia isolates. In addition, 19 different replicon types were determined by PCR-based plasmid replicon typing with IncFII (n=20) being the most common and followed by IncI1α (n=10), IncFIIS (n=8), and IncFIB (n=8). We report, to our knowledge, the first evidence on the presence of FOSr Enterobacterales isolates in raw chicken meat samples in Turkey that might be an important reservoir for FOSr organisms to humans.

Prevalence of fosA3 Gene in Fosfomycin-Resistant Proteus mirabilis in the Autonomous Community of Madrid (Spain) and Characterization of Two Positive Strains.

Plasmid-mediated resistance to fosfomycin has been seldom described in Proteus mirabilis. We report two strains harboring fosA3 gene. Whole-genome sequencing revealed a plasmid that encoded fosA3 gene flanked by two insertion sequence (IS)26 mobile elements. Both strains also produced the blaCTX-M-65 gene that was located in the same plasmid. The sequence detected was IS1182-blaCTX-M-65-orf1-orf2-IS26-IS26-fosA3-orf1-orf2-orf3-IS26. The importance of this transposon lies in its ability to spread in Enterobacterales, therefore, epidemiological surveillance should be carried out.

In Vitro Activity of Fosfomycin on Multidrug-Resistant Strains of Klebsiella pneumoniae and Klebsiella oxytoca Causing Urinary Tract Infection.

With the emergence of multi-drug resistant strains among Klebsiella isolates, the use of old drugs such as fosfomycin has been considered. In this context, we investigated the effect of fosfomycin on biofilm-producing Klebsiella pneumoniae and Klebsiella oxytoca strains isolated from ICU patients. A total of 90 isolates of Klebsiella pneumoniae and 30 isolates of Klebsiella oxytoca were collected from the ICU ward. All isolates were confirmed by biochemical and genotypic methods. Antibiotic susceptibility testing was performed by disc diffusion method and for fosfomycin and colistin, minimum inhibitory concentration (MIC) was done using micro broth dilution. The presence of the beta-lactamase encoding genes, biofilm-related genes, and fosfomycin resistance-related genes was detected by PCR. Finally, for fosfomycin-resistant isolates, we determined the sequence type by the MLST method. Sensitivity rate to fosfomycin in Klebsiella pneumoniae and Klebsiella oxytoca isolates was 92.2% and 100%, respectively. Fosfomycin was the most active antimicrobial agent with 96% sensitivity among all tested antibiotics. All tested isolates could produce biofilm. The frequency of biofilm-related genes for Klebsiella pneumoniae was as follows: 95.5% fimH, 86.6% mrkD, 77.7% mrkA, and 50% wcaG. The frequency of these genes for Klebsiella oxytoca was as follows: 56.6% fimA, 46.6% mrkA, 93.3% matB, and 90% pilQ. Only 4.4% of Klebsiella pneumoniae isolates showed resistance to fosfomycin, and the fosA gene was detected in all of them. Our results showed that fosfomycin effectively inhibits multidrug-resistant (MDR) strains of Klebsiella pneumoniae and Klebsiella oxytoca.

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes.

The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla OKP types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla SHV-1, bla OXA-1, aac(6')-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3")-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIBK was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan.

Genomic insights of Leclercia adecarboxylata strains linked to an outbreak in public hospitals in Mexico.

Leclercia adecarboxylata is a bacteria closely related to Escherichia coli according to its biochemical characteristics and is commonly considered non-pathogenic although a growing number of publications classify it as an emerging pathogen. Fosfomycin resistance is a common trait for L. adecarboxylata encoded by fosALA gene. To analyze genomic traits of sixteen L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition. Twenty-eight L. adecarboxylata strains isolated from blood culture and a bottle of total parenteral nutrition were identified biochemically with a Vitek ® automated system. The strains were phenotyped by their growth on Eosin Methylene Blue agar or MacConkey agar plates. Additionally, Pulsed field gel electrophoresis (PFGE) was performed to establish the clonal relationship. The genomic DNA of sixteen strains was obtained using a Qubit ® dsDNA HS Assay Kit and sequenced on an Illumina ® MiSeq instrument. Draft genomes were assembled using PROKKA and Rast. Assemblies were submitted to Resfinder and PathogenFinder from the Center for Genomic Epidemiology in order to find resistance genes and pathogenic potential. IslandViewer4 was also used to find Pathogenicity and Phage Islands. For identification of the fosA gene, manual curation and Clustal analysis was performed. A novel FosA variant was identified. Finally, phylogenetic analysis was performed using VAMPhyRE software and Mega X. In this paper, we report the genomes of sixteen strains of Leclercia adecarboxylata causing an outbreak associated with parenteral nutrition in public hospitals in Mexico. The genomes were analyzed for genetic determinants of virulence and resistance. A high pathogenic potential (pathogenicity index 0.82) as well as multiple resistance genes including carbapenemics, colistin and efflux pumps were determined. Based on sequence analysis, a new variant of the fosALA gene was described. Finally, the outbreak was confirmed by establishing the clonal relationship among the sixteen genomes obtained. Commensal strains of L. adecarboxylata may acquire genetic determinants that provide mechanisms of host damage and go unnoticed in clinical diagnosis. L. adecarboxylata can evolve in a variety of ways including the acquisition of resistance and virulence genes representing a therapeutic challenge in patient care.

First Report of IMI-2-Producing Enterobacter bugandensis and CTX-M-55-Producing Escherichia coli isolated from Healthy Volunteers in Tunisia.

The aim of this study was to characterize the prevalence of fecal carriage of extended-spectrum beta-lactamases and carbapenemase-producing Gram-negative bacteria among healthy humans in Tunisia. Fifty-one rectal swabs of healthy volunteers were plated on MacConkey agar plates supplemented with cefotaxime or imipenem. The occurrences of resistance genes, integrons, and phylogroup typing were investigated using PCR and sequencing. The genetic relatedness of isolates was determined by pulsed-field-gel-electrophoresis (PFGE) and multilocus-sequence-typing (MLST). Whole-genome-sequencing (WGS) was performed for the carbapenem-resistant isolate. Sixteen ESBL-producing Escherichia coli isolates and one carbapenem-resistant Enterobacter bugandensis were detected out of the fifty-one fecal samples. The ESBL-producing E. coli strains contained genes encoding CTX-M-15 (n = 9), CTX-M-1 (n = 3), CTX-M-27 (n = 3), and CTX-M-55 (n = 1). Three CTX-M-1-producers were of lineages ST131, ST7366, and ST1158; two CTX-M-15-producers belonged to lineage ST925 and ST5100; one CTX-M-27-producer belonged to ST2887, and one CTX-M-15-producer belonged to ST744. Six isolates contained class 1 integrons with the following four gene cassette arrangements: dfrA5 (two isolates), dfrA12-orf-aadA2 (two isolates), dfrA17-aadA5 (one isolate), and aadA1 (one isolate). E. bugandensis belonged to ST1095, produced IMI-2 carbapenemase, and contained qnrE1 and fosA genes. A genome-sequence analysis of the E. bugandensis strain revealed new mutations in the blaACT and qnr genes. Our results reveal an alarming rate of ESBL-E. coli in healthy humans in Tunisia and the first description of IMI-2 in E. bugandensis.

Abstract 2706: AMR Enzymatic inactivation mechanism attributed to fosA Gene antibiotic resistance among Covid 19 hospitalized patients

Abstract 2706: AMR Enzymatic inactivation mechanism attributed to fosA Gene antibiotic resistance among Covid 19 hospitalized patients

Population analysis of heavy metal and biocide resistance genes in Salmonella enterica from human clinical cases in New Hampshire, United States.

Microbes frequently encounter heavy metals and other toxic compounds generated from natural biogeochemical processes and anthropogenic activities. Here, we analyzed the prevalence and association of genes conferring resistance to heavy metals, biocides, and antimicrobial compounds in 394 genome sequences of clinical human-derived S. enterica from New Hampshire, USA. The most prevalent was the gold operon (gesABC-golTSB), which was present in 99.2% of the genomes. In contrast, the other five heavy metal operons (arsenic, copper, mercury, silver, tellurite) were present in 0.76% (3/394)-5.58% (22/394) of the total population. The heavy metal operons and three biocide resistance genes were differentially distributed across 15 sequence types (STs) and 16 serotypes. The number of heavy metal operons and biocide resistance genes per genome was significantly associated with high number of antimicrobial resistance (AMR) genes per genome. Notable is the mercury operon which exhibited significant association with genes conferring resistance to aminoglycosides, cephalosporins, diaminopyrimidine, sulfonamide, and fosfomycin. The mercury operon was co-located with the AMR genes aac(3)-IV, ant(3")-IIa, aph(3')-Ia, and aph(4)-Ia, CTX-M-65, dfrA14, sul1, and fosA3 genes within the same plasmid types. Lastly, we found evidence for negative selection of individual genes of each heavy metal operon and the biocide resistance genes (dN/dS < 1). Our study highlights the need for continued surveillance of S. enterica serotypes that carry those genes that confer resistance to heavy metals and biocides that are often associated with mobile AMR genes. The selective pressures imposed by heavy metals and biocides on S. enterica may contribute to the co-selection and spread of AMR in human infections.

Effect of Colistin, Fosfomycin and Meropenem/Vaborbactam on Carbapenem-Resistant Enterobacterales in Egypt: A Cross-Sectional Study.

The increasing multi-drug carbapenem resistance among Enterobacterales are a severe health problem limiting therapeutic options and worsen the prognosis. This study characterizes carbapenemase genes and integrons among uropathogenic carbapenem resistant Enterobacterales (CRE) isolates recovered from Mansoura University Hospitals and evaluates the effect of colistin, fosfomycin and meropenem-vaborbactam on these isolates. A total of 200 Enterobacterales isolates were collected from patients with urinary tract infections. Antimicrobial susceptibility testing was performed by the disc diffusion method. Colistin susceptibility was tested using the broth microdilution method and fosfomycin and meropenem/vaborbactam susceptibility were tested by MIC Test Strips. Carbapenem resistant isolates were screened for carbapenemase activity phenotypically using the modified carbapenem inactivation method and EDTA-modified carbapenem inactivation method and genotypically by multiplex PCR. Integrons class 1 and 2 and fosA gene were assayed by PCR. Data were statistically analyzed using the Statistical Package for Social Sciences (SPSS) version 16. The Chi-square or Fisher's exact test was used to compare groups, as appropriate. Ninety-two Enterobacterales isolates were resistant to meropenem (46%); 52 E. coli and 40 K. pneumoniae strains. All CRE isolates were multi-drug resistant (MDR). Sensitivity of CRE isolates to colistin, fosfomycin and meropenem/vaborbactam were 67.4%, 82.6% and 58.7%, respectively. Carbapenemase genes were detected by multiplex PCR in 69.6% of CRE isolates (Carbapenemase producing Enterobacterales (CPE) mainly blaNDM (37%). CPE isolates were significantly more resistant to meropenem/vaborbactam than non-CPE isolates; 51.6% vs 17.8%, respectively (P = 0.003) especially blaNDM carrying isolates (70.6%). Class 1 integrons and fosA gene were detected in 91.3% and 11.9% of CRE isolates, respectively. This study revealed that about half of the uropathogenic Enterobacterales isolates were MDR CRE. Carbapenemase gene blaNDM was the main gene among CRE isolates. Meropenem/vaborbactam sensitivity was significantly higher on non-CPE than CPE isolates and limited by the predominance of blaNDM .

Hybrid IncFIA/FIB/FIC(FII) plasmid co-carrying blaNDM-5 and fosA3 from an Escherichia coli ST117 strain of retail chicken

Carbapenems and fosfomycin are important antibiotics used to treat Enterobacteriaceae-associated infections. This study aimed to characterize the co-resistance and co-dissemination mechanism of carbapenem and fosfomycin resistance in an Escherichia coli ST117 strain isolated from retail chicken meat. Antimicrobial susceptibility testing showed that an E. coli CS18F strain had a multidrug resistance profile, including carbapenem and fosfomycin resistance. The presence of blaNDM-5 and fosA3 genes was confirmed by PCR and Sanger sequencing. The blaNDM-5 and fosA3 genes were successfully transferred to the recipient strain E. coli J53 via conjugation, and the transconjugants had elevated minimum inhibitory concentrations (MICs) for meropenem and fosfomycin. Whole genome sequencing (WGS) of E. coli CS18F revealed that blaNDM-5 and fosA3 were colocalized on an IncFIA/FIB/FIC(FII) type plasmid of 189,141 bp, which was designated as pCS18F-NDM-Fos. A novel structure with five IS26 sequences flanking the multiple drug resistance region (MDRR) was identified, and three copies of IS26 were found to be flanked blaNDM-5, fosA3, dfrA12, aadA2, and sul1. Three types of translocation units (TUs) were identified by PCR, containing either the resistance gene blaNDM-5 and an IS26 sequence, fosA3, and an IS26 sequence, or both, indicating their potential co-transfer via TUs. Thus, this is an unprecedented report of the presence of a plasmid co-carrying blaNDM-5 and fosA3 and TUs potentially mediating their simultaneous transfer.

Resensitization of Fosfomycin-Resistant Escherichia coli Using the CRISPR System.

Antimicrobial resistance is a public health burden with worldwide impacts and was recently identified as one of the major causes of death in 2019. Fosfomycin is an antibiotic commonly used to treat urinary tract infections, and resistance to it in Enterobacteriaceae is mainly due to the metalloenzyme FosA3 encoded by the fosA3 gene. In this work, we adapted a CRISPR-Cas9 system named pRE-FOSA3 to restore the sensitivity of a fosA3+ Escherichia coli strain. The fosA3+ E. coli strain was generated by transforming synthetic fosA3 into a nonpathogenic E. coli TOP10. To mediate the fosA3 disruption, two guide RNAs (gRNAs) were selected that used conserved regions within the fosA3 sequence of more than 700 fosA3+ E. coli isolates, and the resensitization plasmid pRE-FOSA3 was assembled by cloning the gRNA into pCas9. gRNA_195 exhibited 100% efficiency in resensitizing the bacteria to fosfomycin. Additionally, the edited strain lost the ampicillin resistance encoded in the same plasmid containing the synthetic fosA3 gene, despite not being the CRISPR-Cas9 target, indicating plasmid clearance. The in vitro analysis presented here points to a path that can be explored to assist the development of effective alternative methods of treatment against fosA3+ bacteria.

Genetic characterization of hypervirulent Klebsiella pneumoniae responsible for acute death in captive marmosets.

Klebsiella pneumoniae is a Gram-negative bacterium implicated as the causative pathogen in several medical health issues with different strains causing different pathologies including pneumonia, bloodstream infections, meningitis and infections from wounds or surgery. In this study, four captive African marmosets housed in Thailand were found dead. Necropsy and histology revealed congestion of hearts, kidneys and adrenal glands. Twenty-four bacterial isolates were obtained from these four animals with all isolates yielding identical phenotypes indicative of K. pneumoniae based on classical identification schema. All the isolates show the susceptibility to amikacin, cephalexin, doxycycline, gentamicin, and enrofloxacin with intermediate susceptibility to amoxycillin/clavulanic acid. One isolate (20P167W) was chosen for genome analysis and determined to belong to sequence type 65 (ST65). The genome of 20P167W possessed multiple virulence genes including mrk gene cluster and iro and iuc gene cluster (salmochelin and aerobactin, respectively) as well as multiple antibiotic resistance genes including blaSHV−67, blaSHV−11, oqxA, oqxB, and fosA genes resembling those found in human isolates; this isolate has a close genetic relationship with isolates from humans in Ireland, but not from Thailand and California sea lions. Phylogenetic studies using SNP show that there was no relation between genetic and geographic distributions of all known strains typing ST65, suggesting that ST65 strains may spread worldwide through multiple international transmission events rather than by local expansions in humans and/or animals. We also predict that K. pneumoniae ST65 has an ability to acquire genetic mobile element from other bacteria, which would allow Klebsiella to become an even greater public health concern.

Molecular characterization of extended-spectrum ß-lactamase producers, carbapenemase producers, polymyxin-resistant, and fosfomycin-resistant Enterobacterales among pigs from Egypt

ObjectivesTo perform the first prospective surveillance evaluating the occurrence of genes encoding colistin resistance, fosfomycin resistance, carbapenemase, or extended-spectrum ß-lactamases (ESBLs) among Enterobacterial isolates recovered from the gut flora of pigs from Egypt. MethodsBetween February and April 2020, 81 rectal swabs were collected from pigs in a slaughterhouse, Cairo, Egypt. Samples were screened for different resistance mechanisms using SuperPolymyxin, ChromID ESBL, SuperFOS, and SuperCarba selective agar plates. Antimicrobial susceptibility testing was performed for all isolates using disk diffusion and broth microdilution techniques. PCR screening was performed for ESBLs, carbapenemases, mcr, and fosA genes. Mating-out assays, multilocus sequence typing analysis, and plasmid typing were also performed. ResultsA high prevalence of ESBLs, carbapenemases, fosfomycin, and colistin resistance genes was evidenced among those isolates. The predominant ESBL identified was blaCTX-M-15, followed by blaCTX-M-9. We also identified blaNDM-5 and blaOXA-244. fosA3, fosA4, and fosA6 were identified in E. coli isolates. In addition, 11 MCR-1 producers were recovered. Notably, co-occurrence of ESBL genes and mcr or fosA genes was observed. MLST analysis revealed a high clonal diversity, ruling out the dissemination of one major clone. IncFIB-type was predominantly present among ESBL and FosA producers. The blaNDM-5 gene was carried on an IncX4-type, although the blaOXA-244 gene was chromosomally located. The mcr-1 gene was carried on a diversity of plasmids (IncI2, IncX4, and IncHI2). ConclusionThese results raise serious public health concerns as Egyptian pig meat could serve as a reservoir for antimicrobial resistance genes (ARGs), leading to worldwide dissemination.

High-Frequency Detection of fosA3 and bla CTX-M-55 Genes in Escherichia coli From Longitudinal Monitoring in Broiler Chicken Farms.

Considering the worrying emergence of multidrug resistance, including in animal husbandry and especially in food-producing animals, the need to detect antimicrobial resistance strains in poultry environments is relevant, mainly considering a One Health approach. Thus, this study aimed to conduct longitudinal monitoring of antimicrobial resistance in broiler chicken farms, with an emphasis on evaluating the frequency of resistance to fosfomycin and β-lactams. Escherichia coli was isolated from broiler chicken farms (cloacal swabs, meconium, poultry feed, water, poultry litter, and Alphitobius diaperinus) in northern Paraná from 2019 to 2020 during three periods: the first period (1st days of life), the second period (20th to 25th days of life), and third period (40th to 42nd days of life). Antibiogram tests and the detection of phenotypic extended-spectrum β-lactamase (ESBL) were performed, and they were confirmed by seaching for genes from the blaCTX–M group. The other resistance genes searched were mcr-1 and fosA3. Some ESBL blaCTX–M–1 group strains were selected for ESBL identification by sequencing and enterobacterial repetitive intergenic consensus-polymerase chain reaction analysis. To determine the transferability of the blaCTX–M–1– and fosA3-carrying plasmids, strains were subjected to conjugation experiments. A total of 507 E. coli were analyzed: 360 from cloacal swabs, 24 from meconium samples, 3 from poultry feed samples, 18 from water samples, 69 from poultry litter samples, and 33 from A. diaperinus samples. Among the strain isolate, 80% (406/507) were multidrug-resistant (MDR), and 51% (260/507) were ESBL-positive, with the blaCTX–M–1 group being the most frequent. For the fosA3 gene, 68% (344/507) of the strains isolated were positive, deserves to be highlighted E. coli isolated from day-old chickens (OR 6.34, CI 2.34–17.17), when compared with strains isolated from other origins (poultry litter, A. diaperinus, water, and poultry feed). This work alerts us to the high frequency of the fosA3 gene correlated with the CTX-M-1 group (OR 3.57, CI 95% 2.7–4.72, p < 0.05), especially the blaCTX–M–55 gene, in broiler chickens. This profile was observed mainly in day-old chicken, with a high percentage of E. coli that were MDR. The findings emphasize the importance of conducting longitudinal monitoring to detect the primary risk points during poultry production.

The Co-occurrence of mcr-3 and fosA3 in IncP plasmid in ST131 Escherichia coli: A novel case.

Plasmid-mediated colistin resistance genes, especially mcr-3 combined with the fosfomycin resistance gene fosA3, are a grave health concern. Our study was designed to determine the epidemiological characteristics of the combination of mcr-3 and fosA3 in Anhui province, China. A total of 127 multi-drug-resistant (MDR) E. coli strains were assessed for antibiotic resistance/sensitivity to detect mcr-3 and fosA3 using polymerase chain reaction (PCR) and sequencing. The genes of interest were conjugated using EC600, and replicon and sequence types (STs) were identified by PCR-based replicon typing (PBRT) and multilocus sequence typing (MLST). Cluster similarity and genomic relatedness among the positive isolates were confirmed by Xbal PFGE. The processed E. coli isolates were highly resistant to the tested antibiotics; the prevalence of mcr-3 was 0.78% in the transferable IncP-type plasmid in ST131, whereas fosA3 prevalence was 38.58% among different transferable plasmids, including IncFIIK, IncFII and IncA/C, and in various STs including ST69, ST1193, ST12, ST46, ST57, ST1196, ST38, ST95, ST131, ST7584 and ST10184. Both were successfully transferred to EC600. The Xbal PFGE cluster exposed similarities among the STs. Our results show that to control the spread of colistin and fosfomycin resistance genes in human pathogens, the ban on colistin must be continued in animal feeding farms not only in China but around the world; additionally, awareness platforms on the use of colistin must be implemented and strict policies in poultry and pig farms must be maintained. Furthermore, fosfomycin misuse by patients and overuse by physicians must be strictly managed to stop the spread of fosfomycin resistance.

WGS-Based Phenotyping and Molecular Characterization of the Resistome, Virulome and Plasmid Replicons in Klebsiella pneumoniae Isolates from Powdered Milk Produced in Germany.

The emergence of Klebsiella pneumoniae (K. pneumoniae) in German healthcare is worrying. It is not well-investigated in the veterinary world and food chains. In the current study, antibiotic susceptibility profiles of 24 K. pneumoniae strains isolated from powdered milk samples produced in Germany were investigated by a microdilution test. Next-generation sequencing (NGS) was applied to identify genomic determinants for antimicrobial resistance (AMR), virulence-associated genes and plasmids replicons. All isolates were susceptible to the majority (14/18) of tested antibiotics. Resistance to colistin, fosfomycin, chloramphenicol and piperacillin was found. The ambler class A ß-lactamase, blaSHV variants were identified in all isolates, of which blaSHV-187 was most prevalent and found in 50% of isolates. Single-nucleotide-variants of oqxA and oqxB conferring resistance to phenicol/quinolone were found in all isolates, and the oqxB17 was the most prevalent found in 46% of isolates. 67% of isolates harbored fosA genes; however, only one was fosfomycin-resistant. Two isolates harbored genes conferring resistance to colistin, despite being susceptible. The majority of identified virulome genes were iron uptake siderophores. Two enterobactins (entB, fepC), six adherence-related genes belonging to E. coli common pilus (ECP) and one secretion system (ompA gene) were found in all isolates. In contrast, yersiniabactin was found in two isolates. One ST23 strain was susceptible to all tested antibiotics, and harbored determinants discriminatory for hypervirulent strains, e.g., aerobactin, salmochelin, yersiniabactin, enterobactin and regulator of mucoid phenotype A genes that are highly associated with hypervirulent K. pneumoniae. The IncF plasmid family was found in all strains, while almost half of the isolates harbored Col440I-type plasmids and nine isolates harbored various Inc-type plasmids. The presence of K. pneumoniae carrying different resistomes and major virulent specific virulomes in powdered milk samples is alarming. This could threaten public health, particularly of neonates and infants consuming dried milk.