Novel Trimethoprim Resistance Gene dfrA49 Identified in Riemerella anatipestifer from China.

Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. A total of 139 Riemerella anatipestifer isolates were collected from different farms in China during 2014 to 2020. Whole genome sequencing (WGS) and genome analysis of R. anatipestifer isolates revealed a 504-bp open reading frame (ORF) encoding a putative dfrA gene. This DfrA variant shared 66.47% amino acid sequence identity with DfrA36 and shared ≤51.20% identity with any other previously identified DfrA proteins. The novel dfrA gene, designated dfrA49, conferred trimethoprim (TMP) resistance when cloned into Escherichia coli BL21(DE3). Thirty dfrA49-positive isolates were identified from Jiangsu and Guangdong province (5/38, 13.16%, and 25/101, 24.75%, respectively). Five of the 38 isolates had obtained the complete genome sequences. Genomic analysis showed that the dfrA49 gene was located on chromosomes or a plasmid (four of them were on chromosomes and one was located on a plasmid). The plasmid p20190305E2-2_2 carried dfrA49, catB, ermF, ereD, blaOXA (88.36% identity with blaOXA-209), Δarr, and tet(X18). Further research indicated that dfrA49 usually coexisted with catB in R. anatipestifer. In this study, a novel trimethoprim resistance gene, dfrA49, was identified and characterized in chromosome and plasmid sequences from R. anatipestifer using WGS and bioinformatic methods. It further expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and provides information about antibiotic resistance genes in R. anatipestifer, where the resistance gene pool circulating is not well understood. IMPORTANCE Trimethoprim is a synthetic antimicrobial agent inhibiting dihydrofolate reductase (DHFR), which is encoded by the folA gene. Acquired genes that confer trimethoprim resistance due to mutations in the folA gene are designated dfr and divided into two main families including dfrA and dfrB. Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. R. anatipestifer belongs to the Flavobacteriaceae family, and the reservoir of dfrA resistance genes in R. anatipestifer has not been fully investigated. A novel trimethoprim resistance gene, dfrA49, which was identified and characterized in chromosome and plasmid sequences in this study, increased the MIC of TMP (>256-fold) in E. coli BL21(DE3). Our study expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and broadens the understanding of the host spectrum of dfrA family genes.

Antimicrobial resistance in urinary pathogens and culture-independent detection of trimethoprim resistance in urine from patients with urinary tract infection.

BACKGROUND: Although urinary tract infections (UTIs) are extremely common, isolation of causative uropathogens is not always routinely performed, with antibiotics frequently prescribed empirically. This study determined the susceptibility of urinary isolates from two Health and Social Care Trusts (HSCTs) in Northern Ireland to a range of antibiotics commonly used in the treatment of UTIs. Furthermore, we determined if detection of trimethoprim resistance genes (dfrA) could be used as a potential biomarker for rapid detection of phenotypic trimethoprim resistance in urinary pathogens and from urine without culture. METHODS: Susceptibility of E. coli and Klebsiella spp. isolates (n = 124) to trimethoprim, amoxicillin, ceftazidime, ciprofloxacin, co-amoxiclav and nitrofurantoin in addition to susceptibility of Proteus mirabilis (n = 61) and Staphylococcus saprophyticus (n = 17) to trimethoprim was determined by ETEST® and interpreted according to EUCAST breakpoints. PCR was used to detect dfrA genes in bacterial isolates (n = 202) and urine samples(n = 94). RESULTS: Resistance to trimethoprim was observed in 37/124 (29.8%) E. coli and Klebsiella spp. isolates with an MIC90 > 32 mg/L. DfrA genes were detected in 29/37 (78.4%) trimethoprim-resistant isolates. Detection of dfrA was highly sensitive (93.6%) and specific (91.4%) in predicting phenotypic trimethoprim resistance among E. coli and Klebsiella spp. isolates. The dfrA genes analysed were detected using a culture-independent PCR method in 16/94 (17%) urine samples. Phenotypic trimethoprim resistance was apparent in isolates cultured from 15/16 (94%) dfrA-positive urine samples. There was a significant association (P < 0.0001) between the presence of dfrA and trimethoprim resistance in urine samples containing Gram-negative bacteria (Sensitivity = 75%; Specificity = 96.9%; PPV = 93.8%; NPV = 86.1%). CONCLUSIONS: This study demonstrates that molecular detection of dfrA genes is a good indicator of trimethoprim resistance without the need for culture and susceptibility testing.

Antimicrobial Resistance of Acetobacter and Komagataeibacter Species Originating from Vinegars.

Consumers' preference towards healthy and novel foods dictates the production of organic unfiltered bottled vinegar that still contains acetic acid bacteria. After ingesting vinegar, the bacteria come into close contact with the human microbiota, creating the possibility of horizontal gene transfer, including genetic determinants for antibiotic resistance. Due to the global spread of antimicrobial resistance (AMR), we analyzed the AMR of Acetobacter and Komagataeibacter species originating mainly from vinegars. Six antibiotics from different structural groups and mechanisms of action were selected for testing. The AMR was assessed with the disk diffusion method using various growth media. Although the number of resistant strains differed among the growth media, 97.4%, 74.4%, 56.4%, and 33.3% of strains were resistant to trimethoprim, erythromycin, ciprofloxacin, and chloramphenicol, respectively, on all three media. Moreover, 17.9% and 53.8% of all strains were resistant to four and three antibiotics of different antimicrobial classes, respectively. We then looked for antimicrobial resistance genes in the genome sequences of the reference strains. The most common genetic determinant potentially involved in AMR encodes an efflux pump. Since these genes pass through the gastrointestinal tract and may be transferred to human microbiota, further experiments are needed to analyze the probability of this scenario in more detail.

MgrB Inactivation Confers Trimethoprim Resistance in Escherichia coli.

After several decades of use, trimethoprim (TMP) remains one of the key access antimicrobial drugs listed by the World Health Organization. To circumvent the problem of trimethoprim resistance worldwide, a better understanding of drug-resistance mechanisms is required. In this study, we screened the single-gene knockout library of Escherichia coli, and identified mgrB and other several genes involved in trimethoprim resistance. Subsequent comparative transcriptional analysis between ΔmgrB and the wild-type strain showed that expression levels of phoP, phoQ, and folA were significantly upregulated in ΔmgrB. Further deleting phoP or phoQ could partially restore trimethoprim sensitivity to ΔmgrB, and co-overexpression of phoP/Q caused TMP resistance, suggesting the involvement of PhoP/Q in trimethoprim resistance. Correspondingly, MgrB and PhoP were shown to be able to modulated folA expression in vivo. After that, efforts were made to test if PhoP could directly modulate the expression of folA. Though phosphorylated PhoP could bind to the promotor region of folA in vitro, the former only provided a weak protection on the latter as shown by the DNA footprinting assay. In addition, deleting the deduced PhoP box in ΔmgrB could only slightly reverse the TMP resistance phenotype, suggesting that it is less likely for PhoP to directly modulate the transcription of folA. Taken together, our data suggested that, in E. coli, MgrB affects susceptibility to trimethoprim by modulating the expression of folA with the involvement of PhoP/Q. This work broadens our understanding of the regulation of folate metabolism and the mechanisms of TMP resistance in bacteria.

Novel Soil-Derived Beta-Lactam, Chloramphenicol, Fosfomycin and Trimethoprim Resistance Genes Revealed by Functional Metagenomics.

Antibiotic resistance genes (ARGs) in soil are considered to represent one of the largest environmental resistomes on our planet. As these genes can potentially be disseminated among microorganisms via horizontal gene transfer (HGT) and in some cases are acquired by clinical pathogens, knowledge about their diversity, mobility and encoded resistance spectra gained increasing public attention. This knowledge offers opportunities with respect to improved risk prediction and development of strategies to tackle antibiotic resistance, and might help to direct the design of novel antibiotics, before further resistances reach hospital settings or the animal sector. Here, metagenomic libraries, which comprise genes of cultivated microorganisms, but, importantly, also those carried by the uncultured microbial majority, were screened for novel ARGs from forest and grassland soils. We detected three new beta-lactam, a so far unknown chloramphenicol, a novel fosfomycin, as well as three previously undiscovered trimethoprim resistance genes. These ARGs were derived from phylogenetically diverse soil bacteria and predicted to encode antibiotic inactivation, antibiotic efflux, or alternative variants of target enzymes. Moreover, deduced gene products show a minimum identity of ~21% to reference database entries and confer high-level resistance. This highlights the vast potential of functional metagenomics for the discovery of novel ARGs from soil ecosystems.

Capture of a novel, antibiotic resistance encoding, mobile genetic element from Escherichia coli using a new entrapment vector.

AIMS: Antimicrobial resistance genes (ARGs) are often associated with mobile genetic elements (MGEs), which facilitate their movement within and between bacterial populations. Detection of mobility is therefore important to understand the dynamics of MGE dissemination and their associated genes, especially in resistant clinical isolates that often have multiple ARGs associated with MGEs. Therefore, this study aimed to develop an entrapment vector to capture active MGEs and ARGs in clinical isolates of Escherichia coli. METHODS AND RESULTS: We engineered an entrapment vector, called pBACpAK, to capture MGEs in clinical E. coli isolates. It contains a cI-tetA positive selection cartridge in which the cI gene encodes a repressor that inhibits the expression of tetA. Therefore, any disruption of cI, for example, by insertion of a MGE, will allow tetA to be expressed and result in a selectable tetracycline-resistant phenotype. The pBACpAK was introduced into clinical E. coli isolates and grown on tetracycline-containing agar to select for clones with the insertion of MGEs into the entrapment vector. Several insertion sequences were detected within pBACpAK, including IS26, IS903B and ISSbo1. A novel translocatable unit (TU), containing IS26 and dfrA8 was also captured, and dfrA8 was shown to confer trimethoprim resistance when it was cloned into E. coli DH5α. CONCLUSIONS: The entrapment vector, pBACpAK was developed and shown to be able to capture MGEs and their associated ARGs from clinical E. coli isolates. We have captured, for the first time, a TU encoding antibiotic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first time that a TU and associated resistance gene has been captured from clinical E. coli isolates using an entrapment vector. The pBACpAK has the potential to be used not only as a tool to capture MGEs in clinical E. coli isolates, but also to study dynamics, frequency and potentiators of mobility for MGEs.

Integrative Analysis of Whole Genome Sequencing and Phenotypic Resistance Toward Prediction of Trimethoprim-Sulfamethoxazole Resistance in Staphylococcus aureus.

As whole genome sequencing is becoming more accessible and affordable for clinical microbiological diagnostics, the reliability of genotypic antimicrobial resistance (AMR) prediction from sequencing data is an important issue to address. Computational AMR prediction can be performed at multiple levels. The first-level approach, such as simple AMR search relies heavily on the quality of the information fed into the database. However, AMR due to mutations are often undetected, since this is not included in the database or poorly documented. Using co-trimoxazole (trimethoprim-sulfamethoxazole) resistance in Staphylococcus aureus, we compared single-level and multi-level analysis to investigate the strengths and weaknesses of both approaches. The results revealed that a single mutation in the AMR gene on the nucleotide level may produce false positive results, which could have been detected if protein sequence analysis would have been performed. For AMR predictions based on chromosomal mutations, such as the folP gene of S. aureus, natural genetic variations should be taken into account to differentiate between variants linked to genetic lineage (MLST) and not over-estimate the potential resistant variants. Our study showed that careful analysis of the whole genome data and additional criterion such as lineage-independent mutations may be useful for identification of mutations leading to phenotypic resistance. Furthermore, the creation of reliable database for point mutations is needed to fully automatized AMR prediction.

Use of other antimicrobial drugs is associated with trimethoprim resistance in patients with urinary tract infections caused by E. coli.

In recent years, high frequencies of trimethoprim resistance in urinary tract infections (UTIs) caused by E. coli are have been reported. Co-resistance to other antimicrobial drugs may play a role in this increase. Therefore, we investigated whether previous use of other antimicrobial drugs was associated with trimethoprim resistance. We conducted a nested case-control study with urinary cultures with E. coli from participants of the Rotterdam Study sent in by general practitioners to the regional laboratory between 1 January 2000 and 1 April 2016. Multivariable logistic regression analysis was performed to study the association between prior prescriptions of several antimicrobial drug groups and trimethoprim resistance using individual participant data. Urinary cultures of 1264 individuals with a UTI caused by E. coli were included. When adjusted for previous other antimicrobial drug use, a history of > 3 prescriptions of extended-spectrum penicillins (OR 1.68; 95% CI 1.10-2.55) was significantly associated with trimethoprim resistance of E. coli as was the use of > 3 prescriptions of sulfonamides and trimethoprim (OR 2.22; 95% CI 1.51-3.26). The use of > 3 prescriptions of nitrofuran derivatives was associated with a lower frequency of trimethoprim resistance (OR 0.60; 95% CI 0.39-0.92), after adjustment for other antimicrobial drug prescriptions. We found that previous use of extended-spectrum penicillins is associated with trimethoprim resistance. On the contrary, previous nitrofurantoin use was associated with a lower frequency of trimethoprim resistance. Especially in individuals with recurrent UTI, co-resistance should be taken into account and susceptibility testing before starting trimethoprim should be considered.

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance.

Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.

Emergence of Plasmid-Borne dfrA14 Trimethoprim Resistance Gene in Shigella sonnei.

The most common mechanism of trimethoprim (TMP)-resistance is the acquisition of dihydrofolate reductase enzyme resistant to this drug. Previous molecular characterization of TMP-genes resistance in Chilean isolates of Shigella sonnei searching for dfrA1 and dfrA8, showed solely the presence of dfrA8 (formerly dhfrIIIc). However, these genetic markers were absent in S. sonnei strains further isolated during an outbreak in 2009. To identify the TMP-resistance gene in these strains, a genomic DNA library from a TMP-resistant (TMP(R)) S. sonnei representative strain for the outbreak was used to clone, select and identify a TMP-resistance marker. The TMP(R) clone was sequenced by primer walking, identifying the presence of the dfrA14 gene in the sul2-strA'-dfrA14-'strA-strB gene arrangement, harbored in a native 6779-bp plasmid. The same plasmid was isolated by transforming with a ~4.2 MDa plasmid extracted from several TMP(R) S. sonnei strains into Escherichia coli. This plasmid, named pABC-3, was present only in dfrA14-positive strains and was homologous to a previously described pCERC-1, but different due to the absence of an 11-bp repetitive unit. The distribution of dfrA1, dfrA8, and dfrA14 TMP-resistance genes was determined in 126 TMP(R) S. sonnei isolates. Most of the strains (96%) carried only one of the three TMP-resistance genes assessed. Thus, all strains obtained during the 2009-outbreak harbored only dfrA14, whereas, dfrA8 was the most abundant gene marker before outbreak and, after the outbreak dfrA1 seems have appeared in circulating strains. According to PFGE, dfrA14-positive strains were clustered in a genetically related group including some dfrA1- and dfrA8-positive strains; meanwhile other genetic group included most of the dfrA8-positive strains. This distribution also correlated with the isolation period, showing a dynamics of trimethoprim genetic markers prevalent in Chilean S. sonnei strains. To our knowledge, dfrA14 gene associated to a small non-conjugative plasmid was detected for the first time in Shigella. Apparently, the strain causing the outbreak must have been introduced, changing drastically the genetic distribution of trimethoprim resistance in Chilean S. sonnei strains.

Antibiotic susceptibility of sulfamethoxazole-trimethoprim resistant Stenotrophomonas maltophilia strains isolated at a tertiary care centre in Hungary.

Sulfamethoxazole-trimethoprim (SXT) is the drug-of-choice in Stenotrophomonas maltophilia caused infections. There has been an increase in resistance to SXT of S. maltophilia over recent years. In this study 30 S. maltophilia clinical isolates resistant to SXT were investigated. Antibiotic susceptibilities for ciprofloxacin, moxifloxacin, levofloxacin, doxycycline, tigecycline, ceftazidime, colistin and chloramphenicol were determined by broth microdilution method. None of the strains were susceptible to ciprofloxacin, tigecycline, ceftazidime or colistin. Only 37% of the isolates were susceptible to levofloxacin or moxifloxacin. Two isolates resistant to all tested antibiotic agents and two others susceptible only to doxycycline were further investigated: susceptibility for combinations of antibiotics was analyzed by checkerboard technique. According to the fractional inhibitory concentration indices calculated, moxifloxacin plus ceftazidime combination was found to be synergistic in each case. Genetic testing revealed the predominance of sul1 gene. Our study concluded that the range of effective antibiotic agents is even more limited in infections caused by SXT-resistant S. maltophilia. In these cases, in vitro synergistic antibiotic combinations could be potential therapeutic options.

Asymmetric mutations in the tetrameric R67 dihydrofolate reductase reveal high tolerance to active-site substitutions.

Type II R67 dihydrofolate reductase (DHFR) is a bacterial plasmid-encoded enzyme that is intrinsically resistant to the widely-administered antibiotic trimethoprim. R67 DHFR is genetically and structurally unrelated to E. coli chromosomal DHFR and has an unusual architecture, in that four identical protomers form a single symmetrical active site tunnel that allows only one substrate binding/catalytic event at any given time. As a result, substitution of an active-site residue has as many as four distinct consequences on catalysis, constituting an atypical model of enzyme evolution. Although we previously demonstrated that no single residue of the native active site is indispensable for function, library selection here revealed a strong bias toward maintenance of two native protomers per mutated tetramer. A variety of such "half-native" tetramers were shown to procure native-like catalytic activity, with similar KM values but kcat values 5- to 33-fold lower, illustrating a high tolerance for active-site substitutions. The selected variants showed a reduced thermal stability (Tm ∼12°C lower), which appears to result from looser association of the protomers, but generally showed a marked increase in resilience to heat denaturation, recovering activity to a significantly greater extent than the variant with no active-site substitutions. Our results suggest that the presence of two native protomers in the R67 DHFR tetramer is sufficient to provide native-like catalytic rate and thus ensure cellular proliferation.

pDB2011, a 7.6 kb multidrug resistance plasmid from Listeria innocua replicating in Gram-positive and Gram-negative hosts.

pDB2011, a multidrug resistance plasmid isolated from the foodborne Listeria innocua strain TTS-2011 was sequenced and characterized. Sequence analysis revealed that pDB2011 had a length of 7641 bp and contained seven coding DNA sequences of which two were annotated as replication proteins, one as a recombination/mobilization protein and one as a transposase. Furthermore, pDB2011 harbored the trimethoprim, spectinomycin and macrolide-lincosamide-streptogramin B resistance genes dfrD, spc and erm(A), respectively. However, pDB2011 was only associated with trimethoprim and spectinomycin resistance phenotypes and not with phenotypic resistance to erythromycin. A region of the plasmid encoding the resistance genes spc and erm(A) plus the transposase was highly similar to Staphylococcus aureus transposon Tn554. The dfrD gene was 100% identical to dfrD found in a number of Listeria monocytogenes isolates. Additionally, assessment of the potential host range of pDB2011 revealed that the plasmid was able to replicate in Lactococcus lactis subsp. cremoris MG1363 as well as in Escherichia coli MC1061 and DH5α. This study reports the first multidrug resistance plasmid in L. innocua. A large potential for dissemination of pDB2011 is indicated by its host range of both Gram-positive and Gram-negative bacteria.

Cotrimoxazole-resistant Escherichia coli bacteremia in neutropenic patients at a regional oncology hospital.

In a regional oncology hospital using cotrimoxazole (trimethoprim-sulphamethoxazole) prophylaxis during chemotherapy-induced neutropenia, a single strain of Escherichia coli (indole negative) caused 15 of 27 episodes of Gram-negative rod bacteremia in 1987, and four of 32 such episodes in 1988. This biotype had not been recovered in 1986. Investigations during this 'outbreak' of bacteremias revealed enteric colonization with this strain of E coli in 37% of patients on leukemia or bone marrow transplant wards and in several staff members in July 1987. In 1988, 11 of 32 Gram-negative rod bacteremias were secondary to other strains of indole positive E coli of several different biotypes and plasmid profiles. Indole negative strains all exhibited low level trimethoprim resistance, whereas indole positive strains which subsequently appeared exhibited high level trimethoprim resistance. Failure of cotrimoxazole prophylaxis was initially due to the clonal dissemination of a single strain of E coli within the institution, with the subsequent appearance of multiple E coli strains with probable differing genetic bases for their resistance.