Quinolone Resistance-determining Region Research Articles

Salmonella Typhi Haplotype 58 biofilm formation and genetic variation in isolates from typhoid fever patients with gallstones in an endemic setting in Kenya

Although typhoid fever has largely been eliminated in high-income countries, it remains a major global public health concern especially among low- and middle-income countries. The causative agent, Salmonella enterica serovar Typhi (S. Typhi), is a human restricted pathogen with a limited capacity to replicate outside the human host. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen for an ill-defined period of time after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in a typhoid endemic setting in Nairobi, Kenya. Whole-genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool samples, and 2 from blood samples, all genotype 4.3.1 (H58). Out of this, 19 strains were from four patients also diagnosed with gallstones, of whom three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance-determining region (QRDR), and only sub-lineage 4.3.1.2.EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR) isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. For missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes, and genetic variations in S. Typhi during asymptomatic carriage.

Analysis of molecular mechanisms of delafloxacin resistance in Escherichia coli

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

Effect of single parenteral administration of marbofloxacin on bacterial load and selection of resistant Enterobacteriaceae in the fecal microbiota of healthy pigs

BackgroundAntimicrobial resistance (AMR) is a global concern impacting both humans, animals and their environment. The use of oral antimicrobials in livestock, particularly in pigs, has been identified as a driver in the selection of AMR bacteria. The aim of the present study was to evaluate the effects of a single intramuscular (IM) dose of marbofloxacin (8 mg/kg) on Enterobacteriaceae and E. coli populations, as well as on fluoroquinolone resistance within the fecal microbiota of pigs. Twenty healthy pigs, 60-days old, were divided into two groups: a treated group (n = 13) and a control group (n = 7) and were monitored over a 28-day experimental period. Fecal samples were collected from all animals for the isolation of E. coli and Salmonella strains. The minimum inhibitory concentration (MIC) of marbofloxacin for the isolates recovered on MacConkey agar supplemented with 1 or 4 µg/mL of marbofloxacin and for some generic E. coli isolates (recovered from MacConkey agar not supplemented with marbofloxacin) was determined using the broth microdilution method. Genomic DNA was extracted from the confirmed bacterial strains and sequenced using the Sanger method to identify mutations in the quinolone resistance determining regions (QRDRs) of the gyrA and parC genes.ResultsThe single IM administration of marbofloxacin resulted in a significant decrease in Enterobacteriaceae and E. coli fecal populations from days 1 to 3 post- treatment. No Salmonella isolates were detected in either group, and no marbofloxacin-resistant E. coli isolates were identified. The MIC of the selected generic E. coli strains (n = 100) showed an increase to up to 0.5 µg/mL between days 1 and 3 post-treatment but remained below the clinical breakpoint of marbofloxacin resistance (4 µg/mL). Sequencing of these isolates revealed no mutations in gyrA and parC genes.ConclusionsThe present study showed that this dosing regimen of marbofloxacin significantly decreases the fecal shedding of Enterobacteriaceae and E. coli populations in pigs, while limiting the selection of marbofloxacin-resistant E. coli isolates. These findings warrant validation in sick pigs to support the selective use of this antibiotic solely in cases of clinical disease, thereby minimizing the reliance on conventional (metaphylactic) group treatments in pigs.

Genetic and in-silico approaches for investigating the mechanisms of ciprofloxacin resistance in Salmonella typhi: Mutations, extrusion, and antimicrobial resistance

Salmonella enterica serovar Typhi spreads typhoid infection in humans through the consumption of contaminated food and water. Poor sanitation plays a pivotal role in its dissemination. Over time, the bacterium has acquired resistance to many promising antibiotics, posing a growing global health concern and hindering the achievement of sustainable development goals. This study aims to elucidate the molecular complexity of fluoroquinolone resistance, a first-line treatment for typhoid infection. To achieve this aim, 80 clinical isolates were collected from various diagnostic laboratories. These isolates were confirmed based on morphological characteristics and biochemical tests. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were identified using the Kirby-Bauer disc diffusion method. The mechanism of ciprofloxacin resistance was investigated by sequencing the quinolone resistance-determining region (QRDR) genes and identifying the presence of the qnrS1 gene. As a result of this study, 60 % of isolates showed resistance to ciprofloxacin. At the same time, the qnrS1 gene was present in all the selected strains while mutation analysis identified significant mutation in QRDR of DNA gyrase subunit A (gyrA) and Topoisomerase IV (parC) gene. The combinatorial effect was further investigated by downloading 286 draft genomes. The Mutation analysis reveals significant mutations at gyrA S83F, gyrA D87N, gyrA S83Y, gyrB S464F, parC S80I, and parE L416F. Additionally, docking analysis indicates reduced binding affinity and altered solvent accessibility, which show the structural changes at mutation sites. This study provides crucial insights that mutation reduces the binding affinity while qnrS1 acts as a transport channel to extrude the ciprofloxacin. In the future, further validation through experimental mutagenesis is recommended, for targeted therapeutic interventions against the mounting threat of antibiotic-resistant S. Typhi.

Relationship Between Fluoroquinolone Resistance and Mutations in the Quinolone Resistance-Determining Region in Corynebacterium macginleyi.

The purpose of this study was to investigate the bacterial composition in the conjunctiva and to determine the relationship between fluoroquinolone resistance and mutations in the quinolone resistance-determining region (QRDR) in Corynebacterium macginleyi (C. macginleyi). Bacteria isolated from conjunctival swabs of patients awaiting ophthalmic surgery or patients with presumed keratoconjunctivitis were included in this study. For C. macginleyi isolates from 49 samples, the minimum inhibitory concentrations (MICs) of second- to fourth-generation fluoroquinolones were determined by broth microdilution. Additionally, we determined the sequence of the QRDR in the gyrA gene of C. macginleyi-positive isolates by direct sequencing and investigated the relationship between the QRDR mutation and the MICs of fluoroquinolones for C. macginleyi. Among 423 eyes of 296 preoperative patients who underwent conjunctival culture testing, 105 eyes of 89 patients were culture-positive, and among 148 eyes of 147 patients with keratoconjunctivitis, 55 eyes of 54 patients were culture-positive. C. macginleyi accounted for the largest proportion of cultured organisms (34.8%). C. macginleyi-positive isolates were found in 45 eyes of 37 preoperative patients and in 4 eyes of 4 patients with keratoconjunctivitis. Direct sequencing revealed that 91.8% of C. macginleyi-positive isolates had amino acid mutations in the QRDR and 95.5% of mutations were found at Ser-87 and Asp-91. Isolates harboring double mutations at Ser-87 and Asp-91 were resistant to second- to fourth-generation fluoroquinolones. One isolate with double mutations at Ser-87 and Ala-88 but no mutation in Asp-91 showed intermediate susceptibility to moxifloxacin, a fourth-generation fluoroquinolone. C. macginleyi isolated from conjunctiva harboring QRDR amino acid mutations were resistant to second- to fourth-generation fluoroquinolones.

Prevalence and Genomic Characterization of Multidrug-Resistant Salmonella enterica Serovar Kentucky Sequence Type 198 Circulating - Beijing Municipality, China, 2016-2023.

Highly fluoroquinolone-resistant Salmonella enterica serovar Kentucky (S. Kentucky) of sequence type (ST) 198 has emerged as a global multidrug-resistant (MDR) clone, posing a threat to public health. Whole genome sequencing and antibiotic susceptibility testing was used to characterize the population structure and evolutionary history of 54 S. Kentucky isolates recovered from food and human clinical cases in Beijing from 2016 to 2023. All 54 S. Kentucky ST198 isolates exhibited resistance to quinolones, carrying point mutations in the quinolone resistance-determining regions (gyrA_S83F and parC_S80I). Resistance to other antibiotics (folate pathway inhibitors, cephems, aminoglycosides, phenicols, rifamycin, fosfomycin, macrolides, and tetracyclines), mediated by the sul1, sul2, dfrA14, bla CTX-M, bla TEM-1B, aac(3)-Id, aadA2, aadA7, aph(3')-I, aph(3'')-Ib, rmtB, floR, arr-2, fosA, mph(A), and tet(A) genes, was also observed in different combinations. The Beijing S. Kentucky ST198 evolutionary tree was divided into clades 198.2-1 and 198.2-2, which were further differentiated into three subclades: 198.2-2A, 198.2-2B, and 198.2-2C. Compared with the extended-spectrum β-lactamase-encoding gene bla CTX-M-14b in 198.2-1, the co-existence of bla CTX-M-55 and bla TEM-1B, as well as chromosomally located qnrS1, was detected in most 198.2-2 isolates, which showed more complex MDR phenotypes. S. Kentucky ST198 outbreak isolates derived from two predominant clonal sources: 198.2-1 with cgST236434 and 198.2-2A with cgST296405. The S. Kentucky population in Beijing is genetically diverse, consisting of multiple co-circulating lineages that have persisted since 2016. Strengthening surveillance of food and humans will aid in implementing measures to prevent and control the spread of AMR.

Salmonella Typhi genotypic diversity, cluster identification and antimicrobial resistance determinants in Mukuru settlement, Nairobi Kenya

BackgroundUnderstanding the source of typhoid infections and the genetic relatedness of Salmonella Typhi (S. Typhi) by cluster identification in endemic settings is critical for establishing coordinated public health responses for typhoid fever management. This study investigated the genotypic diversity, antibiotic resistance mechanisms, and clustering of 35 S.Typhi strains isolated from cases and carriers in the Mukuru Informal Settlement.MethodsWe studied 35 S.Typhi isolates, including 32 from cases and 3 from carriers, from study participants in the informal settlement of Mukuru, Nairobi, Kenya. Genomic DNA was extracted, and whole-genome sequencing (WGS) was performed to determine the phylogenetic relatedness of strains and detect antimicrobial resistance determinants (AMR). WGS data were analyzed using bioinformatics tools available at the Center for Genomic Epidemiology and Pathogenwatch platforms.ResultsGenotype 4.3.1.2 EA3 was found to be dominant at 46% (16/35), followed by 4.3.1.2 EA2 at 28% (10/35), and 4.3.1.1 EA1 at 27% (9/35). A comparison of the isolates with global strains from Pathogenwatch identified close clustering with strains from Uganda, Tanzania, Rwanda, and India. Three isolates (9%) distributed in each cluster were isolated from carriers. All genotype 4.3.1.2 EA3 isolates were genotypically multidrug-resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Single mutations in the quinolone resistance-determining region were identified in the gyrA (S83Y) and gyrB (S464F) genes. All isolates associated with multidrug resistance showed the presence of the IncQ1 plasmid with the following genes: blaTEM-1B, catA1, sul1, sul2, and dfrA7.ConclusionThe close phylogenetic relatedness between antimicrobial-resistant case isolates and carriage isolates indicates that typhoid carriage is a possible source of infection in the community. Comparative analysis with global isolates revealed that the Kenyan isolates share common lineages with strains from neighboring East African countries and India, suggesting regional dissemination of specific MDR clones. AMR was a major feature of the isolates. Surveillance and testing for antimicrobial susceptibility should inform options for the management of cases.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates

BackgroundThe emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing.ResultsOut of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6’)-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene.ConclusionsOur results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Antimicrobial susceptibility and virulence gene analysis of Shigella species causing dysentery in Iranian children: Implications for fluroquinolone resistance

Shigella species significantly impact global health due to their role in diarrheal diseases. A 2019–2022 cross-sectional study on 432 stool samples from pediatric patients in Mashhad, Iran, identified Shigella spp. and tested their susceptibility to 12 antimicrobials by the disk diffusion method. The presence of virulence factors, namely ipaH, virA, stx1, and stx2, as well as plasmid-mediated quinolone resistance (PMQR) genes, including qnrA, qnrB, qnrC, qnrD, and qnrS, were ascertained through the utilization of polymerase chain reaction techniques. Sequencing of 15 isolates detected mutations within quinolone resistance-determining regions (QRDRs) at the gyrA and parC genes, indicating fluoroquinolone (FQ) resistance. 19.2 % (83/432) of stool samples contained Shigella, primarily S. sonnei (77.1 %), followed by S. flexneri (21.6 %) and S. boydii (1.2 %). Most isolates were from children under five (55.4 %). All strains had the ipaH gene, lacked stx1 and stx2, and 86.7 % had virA. High resistance was noted for ampicillin and tetracycline (84.3 % each), trimethoprim-sulfamethoxazole (81.9 %), and azithromycin (60.2 %). 87.1 % of isolates were multidrug-resistant (MDR). The most common PMQR genes were qnrA and qnrS (41 % each). The qnrD gene, prevalent in 36.1 % of cases, is reported in Iran for the first time. The most common PMQR profile was qnrADS (15.7 %). Resistance to nalidixic acid and ciprofloxacin was 45.8 % and 12 %, respectively. The Shigella isolates exhibited mutations in the gyrA (at codons 83, 87, and 211) and parC (at codons 80, 84, 93, 126, 128, 129, and 132) genes. The D87Y mutation in the gyrA gene was the most common in Shigella isolates, occurring in 73 % of cases. The F93S and L132T mutations in the parC gene were unique to this study. Empirical FQ therapy in patients infected with MDR Shigella, possessing PMQR determinants and/or mutations in the QRDRs of gyrA and parC, may escalate the risks of secondary diseases, extended treatment duration, therapeutic failure, and resistance spread. Consequently, the necessity for continuous surveillance and genetic testing to detect FQ-resistant Shigella strains is of paramount importance.

Prevalence and characterization of foodborne pathogens isolated from fresh-cut fruits and vegetables in Beijing, China

Pre-cut fresh fruits and vegetables are highly appealing to consumers for their convenience, however, as they are highly susceptible to microbial contamination in processing, the potential risks of foodborne illnesses to public health are not negligible. This study aimed to assess the prevalence, antibiotic susceptibility and molecular characteristics of major foodborne pathogens (Listeria monocytogenes, Escherichia coli, Staphylococcus aureus and Salmonella) isolated from fresh-cut fruits and vegetables in Beijing, China. 86 stains were isolated from 326 samples, with S. aureus being the highest prevalence (15.38 %), followed by E. coli (9.23 %) and L. monocytogenes (1.85 %), while no Salmonella was detected. The prevalence by type of food indicated that fruit trays and mixed vegetables were more susceptible to contamination by pathogens. 98 % of S. aureus were resistant to at least of one antibiotic, and showed a high resistance rate to benzylpenicillin (90 %) and oxacillin (48 %). Among 25 E. coli isolates, 57.67 % of which exhibited multi-drug resistance, with common resist to trimethoprim/sulfamethoxazole (66.67 %) and ampicillin (63.33 %). A total of 9 sequence types (STs) and 8 spa types were identified in 35 S. aureus isolates, with ST398-t34 being the predominant type (42.86 %). Additionally, analysis of 25 E. coli isolates demonstrated significant heterogeneity, characterized by 22 serotypes and 18 STs. Genomic analysis revealed that 5 and 44 distinct antibiotic resistance genes (ARGs) in S. aureus and E. coli, respectively. Seven quinolone resistance-determining regions (QRDRs) mutations were identified in E. coli isolates, of which GyrA (S83L) was the most frequently detected. All the S. aureus and E. coli isolates harbored virulence genes. ARGs in S. aureus and E. coli showed a significant positive correlation with plasmids. Furthermore, one L. monocytogenes isolate, which was ST101 and serogroupIIc from watermelon sample, harbored virulence genes (inlA and inlB) and LIPI-1 pathogenic islands (prfA, plcA, hly and actA), which posed potential risks for consumer's health. This study focused on the potential microbial risk of fresh-cut fruits and vegetables associated with foodborne diseases, improving the scientific understanding towards risk assessment related to ready-to-eat foods.

Salmonella Typhi Haplotype 58 (H58) Biofilm Formation and Genetic Variation in Typhoid Fever Patients with Gallstones in an Endemic Setting in Kenya.

The causative agent of typhoid fever, Salmonella enterica serovar Typhi, is a human restricted pathogen. Human carriers, 90% of whom have gallstones in their gallbladder, continue to shed the pathogen after treatment. The genetic mechanisms involved in establishing the carrier state are poorly understood, but S. Typhi is thought to undergo specific genetic changes within the gallbladder as an adaptive mechanism. In the current study, we aimed to identify biofilm forming ability and the genetic differences in longitudinal clinical S. Typhi isolates from asymptomatic carriers with gallstones in Nairobi, Kenya. Whole genome sequences were analyzed from 22 S. Typhi isolates, 20 from stool and 2 from blood samples, all genotype 4.3.1 (H58). Nineteen strains were from four patients also diagnosed with gallstones, of whom, three had typhoid symptoms and continued to shed S. Typhi after treatment. All isolates had point mutations in the quinolone resistance determining region (QRDR) and only sub-lineage 4.3.1.2EA3 encoded multidrug resistance genes. There was no variation in antimicrobial resistance patterns among strains from the same patient/household. Non-multidrug resistant (MDR), isolates formed significantly stronger biofilms in vitro than the MDR isolates, p<0.001. A point mutation within the treB gene (treB A383T) was observed in strains isolated after clinical resolution from patients living in 75% of the households. Missense mutations in Vi capsular polysaccharide genes, tviE P263S was also observed in 18% of the isolates. This study provides insights into the role of typhoid carriage, biofilm formation, AMR genes and genetic variations in S. Typhi from asymptomatic carriers.

Evaluation of genetic correlation with fluoroquinolones resistance in rifampicin-resistant Mycobacterium tuberculosis isolates

ObjectiveTo detect levofloxacin (LFX) and moxifloxacin (MFX) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates, and predict the resistance level based on specific mutations in gyrA and gyrB genes. MethodsA total of 686 RR-TB isolates were collected from Chinese Drug Resistance Surveillance Program from 2013 to 2020. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole genome sequencing (WGS) analysis. ResultsAmong the 686 RR isolates, the most prevalent resistance was to isoniazid (80.5 %) and ethambutol (28.4 %), followed by LFX (26.1 %) and MFX (21.9 %). The resistance rate of LFX (26.1%–99.4 %) was higher than that of MFX (21.9%–83.3 %) across various drug resistance patterns. Of the 180 fluoroquinolones (FQs) resistant isolates, 168 (93.3 %) had mutations in quinolone-resistant determining regions (QRDRs) with 21 mutation types, and Asp94Gly (32.7 %, 55/168) was the predominant mutation. Isolates with mutations in Asp94Asn and Asp94Gly were associated with high levels of resistance to LFX and MFX. Using broth microdilution method as gold standard, the sensitivities of WGS for LFX and MFX were 93.3 % and 98.0 %, and the specificities were 98.6 % and 95.0 %, respectively. ConclusionThe resistance rate of LFX was higher than that of MFX among various drug resistance patterns in RR-TB isolates. The gyrA Asp94Gly was the predominant mutation type underlying FQs resistance. However, no significant difference was observed between mutation patterns in gyrA gene and resistance level of FQs.

Genomic and Functional Characterization of CTX-M-15-Producing Klebsiella pneumoniae ST307 Isolated from Imported Leopard Tortoises in Germany

The Klebsiella pneumoniae ST307 clone, identified in the mid-1990s, has emerged as a global antimicrobial-resistant (AMR) high-risk clone, significantly contributing to the global health challenge also posed by other AMR K. pneumoniae lineages. The acquisition of a blaCTX-M-15-carrying plasmid has facilitated its widespread dissemination. At Europe’s major transport hub for the movement of live animals, Frankfurt Airport, a shipment of 20 live leopard tortoises was sampled during German border control in 2014. Phylogenetic analysis (MLST) identified a K. pneumoniae ST307 strain, prompting further investigation. Our analysis revealed the presence of a ~193 kb plasmid carrying a broad range of AMR genes, including blaCTX-M-15, blaTEM-1B, blaOXA-1, aac(3)-IIa, aac(6′)-Ib-cr, aph(3″)-Ib, aph(6)-Id, and qnrB1. Additionally, mutations in the quinolone resistance-determining region in gyrA (S83I) and parC (S80I) were detected. Phenotypic testing demonstrated resistance of the isolate to the most common antimicrobials used in both human and veterinary medicine; exceptions included carbapenems and newer β-lactamase inhibitor combinations. Because the role of imported exotic animals in the dissemination of AMR genes is largely deficient, the present study fills yet missing mosaic pieces in the complete picture of extended-spectrum β-lactamase (ESBL)-producing Enterobacterales.

Phenotypic and genomic characterization of a multidrug-resistant Salmonella enterica serovar Kentucky ST198 isolated from a patient in China

ObjectivesThe objective of this study was to investigate the resistance mechanisms of a multidrug-resistant Salmonella Kentucky ST198 FJ-2064 isolated from a patient in China. MethodsThe antimicrobial susceptibility of FJ-2064 was determined by the standard disc dilution and broth microdilution methods. The complete genome of FJ-2064 was sequenced using PacBio and Illumina MiSeq platforms. Polymerase chain reaction (PCR) and S1-PFGE were utilized to confirm the mutation sites and the genomic plasmids, respectively. ResultsIsolate FJ-2064 belongs to sequence type ST198 and harboured no visible large plasmids, but was concurrent resistant to 22 detected antimicrobial agents including cefotaxime, ciprofloxacin, and azithromycin. The complete genome sequence identified 20 acquired antibiotic resistance genes (ARGs) and five chromosomal mutations in the gyrA and parC genes of the quinolone resistance determining regions (QRDRs) in FJ-2064. In addition, PCR sequencing confirmed that most of the ARGs were clustered on one multidrug-resistant region and a variant of SGI1-K. In particular, the bla-TEM-1 and bla-CTX−M-55, qnrS1, mph(A) genes, which confer resistance to cephalosporins, quinolones, and macrolides respectively, were all located on the multidrug-resistant region. ConclusionsWe have demonstrated one multidrug-resistant region and a variant of SGI1-K in a Salmonella Kentucky ST198 that is co-resistant to cefotaxime, ciprofloxacin, and azithromycin.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

A novel variant in Salmonella genomic island 1 of multidrug-resistant Salmonella enterica serovar Kentucky ST198.

Salmonella enterica serovar Kentucky ST198 is a major health threat due to its resistance to ciprofloxacin and several other drugs, including third-generation cephalosporins. Many drug-resistant genes have been identified in the Salmonella genomic island 1 variant K (SGI1-K). In this study, we investigated the antimicrobial resistance (AMR) profile and genotypic relatedness of two isolates of ciprofloxacin-resistant (CIPR) S. Kentucky ST198 from poultry in Northeastern Thailand. We successfully assembled the complete genomes of both isolates, namely SSSE-01 and SSSE-03, using hybrid de novo assembly of both short- and long-read sequence data. The complete genomes revealed their highly similar genomic structures and a novel variant of SGI1-K underlying multidrug-resistant (MDR) patterns, including the presence of blaTEM-1b, which confers resistance to beta-lactams, including cephalosporins and lnu(F) which confers resistance to lincomycin and other lincosamides. In addition, the chromosomal mutations in the quinolone resistance-determining region (QRDR) were found at positions 83 (Ser83Phe) and 87 (Asp87Asn) of GyrA and at positions 57 (Thr57Ser) and 80 (Ser80Ile) of ParC suggesting high resistance to ciprofloxacin. We also compared SSSE-01 and SSSE-03 with publicly available complete genome data and revealed significant variations in SGI1-K genetic structures and variable relationships to antibiotic resistance. In comparison to the other isolates, SGI1-K of SSSE-01 and SSSE-03 had a relatively large deletion in the backbone, spanning from S011 (traG∆) to S027 (resG), and the inversion of the IS26-S044∆-yidY segment. Their MDR region was characterized by the inversion of a large segment, including the mer operon and the relocation of IntI1 and several resistance genes downstream of the IS26-S044∆-yidY segment. These structural changes were likely mediated by the recombination of IS26. The findings broaden our understanding of the possible evolution pathway of SGI1-K in fostering drug resistance, which may provide opportunities to control these MDR strains.IMPORTANCEThe emergence of ciprofloxacin-resistant (CIPR) Salmonella Kentucky ST198 globally has raised significant concerns. This study focuses on two poultry isolates from Thailand, revealing a distinct Salmonella genomic island 1 variant K (SGI1-K) genetic structure. Remarkably, multiple antibiotic resistance genes (ARGs) were identified within the SGI1-K as well as other locations in the chromosome, but not in plasmids. Comparing the SGI1-K genetic structures among global and even within-country isolates unveiled substantial variations. Intriguingly, certain isolates lacked ARGs within the SGI1-K, while others had ARGs relocated outside. The presence of chromosomal extended-spectrum β-lactamase (ESBL) genes and lincosamide resistance, lnu(F), gene, could potentially inform the choices of the treatment of CIPRS. Kentucky ST198 infections in humans. This study highlights the importance of understanding the diverse genetic structures of SGI1-K and emphasizes the role of animals and humans in the emergence of antimicrobial resistance.

A Set of Multiresistant Isolates of Mycoplasma bovis Subtype ST-1 with a Variable Susceptibility to Quinolones Are Also Circulating in Spain.

Mycoplasma bovis (M. bovis) is one of the worldwide most important infectious agents involved in respiratory complex diseases (RCD). In Spain, the endemic presence of subtypes ST-2 and ST-3 with phenotypic differences linked to their susceptibility to fluoroquinolones opened the way to develop control strategies focused on previous diagnosis of the subtype and the use of directed therapies when M. bovis were involved in RCD. Surprisingly, microbiological studies conducted during 2023 evidenced for the first time the presence of Spanish isolates of a new polC-subtype, previously classified as ST-1, recovered from calves with respiratory symptoms and pneumonia in different areas of the country (n = 16). Curiously, the minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed phenotypic differences between these ST-1 isolates when using fluoroquinolones (FLQ). There is no geographical correlation between MIC profiles even for a set of 8 isolates recovered from different animals in the same flock. Sequencing of 4 genes (gyrA, gyrB, parC and parE) encoding quinolone resistance-determining regions (QRDR) evidenced the presence of accumulate mutations in 2 ST-1 isolates with high FLQ MICs, but not in all them (n = 3), thus suggesting that, as previously recorded for ST-2 isolates, other mechanisms should be involved in the acquisition of resistence to these antimicrobials. Additionally, as previously detected in the Spanish ST-2 and ST-3, subtype ST-1 isolates are also resistant to macrolides or lincosamides.

Whole genomic sequence of Salmonella Paratyphi B isolated from a hematology patient in the northern region of Brazil

Salmonella Typhi is a zoonotic pathogen responsible for a life-threatening bloodstream infection that is common in places with limited sanitation and hygiene. This study aimed to characterize the resistance genes and sequence type (ST) of Salmonella Paratyphi isolated from a patient with acute myeloid leukemia (AML) undergoing treatment at Fundação HEMOAM, in the north of Brazil. Phenotypic identification and antimicrobial susceptibility testing were performed using the VITEK-2 system. The genomic DNA was sequenced in Illumina® equipment. Resistance genes were identified through the Genome Annotation Service. The microbiological examination of the blood culture identified S. Paratyphi B. Regarding resistance genes, aminoglycoside acetyltransferase [aac(6')-Iaa] mutations in the Gly133Glu position of GyrA and Ile249Met of ParC were identified. Multilocus sequence typing detected the ST 313 sequence type. Salmonella Paratyphi B isolated from the acute myeloid leukemia patient showed cross-resistance with other antibiotics such as quinolones and tetracyclines, which resulted from mutations in the quinolone resistance-determining region and efflux pump systems.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Development of a detection chip for major pathogenic drug-resistant genes and drug targets in bovine respiratory system diseases.

Bovine respiratory disease (BRD) is a significant veterinary challenge, often exacerbated by pathogen resistance, hindering effective treatment. Traditional testing methods for primary pathogens - Mycoplasma bovis, Pasteurella multocida, and Mannheimia haemolytica - are notably time-consuming and lack the rapidity required for effective clinical decision-making. This study introduces a TaqMan MGB probe detection chip, utilizing fluorescent quantitative PCR, targeting key BRD pathogens and associated drug-resistant genes and sites. We developed 94 specific probes and primers, embedded into a detection chip, demonstrating notable specificity, repeatability, and sensitivity, reducing testing time to under 1 h. Additionally, we formulated probes to detect mutations in the quinolone resistance-determining region, associated with fluoroquinolone resistance in BRD pathogens. The chip exhibited robust sensitivity and specificity, enabling rapid detection of drug-resistant mutations in clinical samples. This methodology significantly expedites the diagnostic process for BRD and sensitive drug screening, presenting a practical advancement in the field.