Mutations In parC Research Articles

Prolonged sitafloxacin and doxycycline combination regimen for treating infections by highly resistant Mycoplasma genitalium.

Mycoplasma genitalium, which causes sexually transmitted diseases, is increasingly resistant to key antibiotics such as macrolides and quinolones, posing a challenge for treatment. To assess the effectiveness of prolonged sitafloxacin and doxycycline combination therapy as a new alternative treatment strategy for highly drug-resistant M. genitalium strains. A prospective cohort study was conducted at the National Center for Global Health and Medicine, Tokyo, Japan, from 1 January 2020 to 31 October 2022. Patients with M. genitalium urogenital or rectal infections and those who did not receive the initial sitafloxacin monotherapy were included. Patients were administered sitafloxacin and doxycycline for 21 days as salvage therapy. M. genitalium isolates were tested for parC, gyrA and 23S rRNA resistance-associated mutations. Twenty-seven patients received the combination therapy. All M. genitalium strains available for resistance analysis had parC (24/24) and macrolide resistance-associated (25/25) mutations, and 68% (17/25) had gyrA mutations. The overall cure rate was 77.8%. For strains with concurrent parC and gyrA mutations, the cure rate was 68.8% (P = 0.053) compared with that for monotherapy (37.5%). Prolonged combination therapy is highly effective against M. genitalium strains with concurrent parC and gyrA mutations. Future research should focus on establishing the optimal treatment duration and monitoring the risk of resistance.

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.

Prevalence, antimicrobial resistance, and genomic characterization of Salmonella strains isolated in Hangzhou, China: a two-year study.

This study explored the molecular epidemiology and resistance mechanisms of 271 non-duplicate Salmonella enterica (S. enterica) strains, isolated mainly from adults (209/271) in a tertiary hospital in Hangzhou between 2020 and 2021. Through whole-genome sequencing and bioinformatics, the bacterial strains were classified into 46 serotypes and 54 sequence types (ST), with S. Enteritidis, S. 1,4,[5],12:i:-, and S. Typhimurium being the most prevalent serotypes and ST11, ST34, and ST19 the most common STs. The strains isolated from adults were primarily S. Enteritidis (59/209), while from children were mainly S. 1,4,[5],12:i:- (20/62). Worryingly, 12.55% strains were multi-drug resistant (MDR), with resistance rates to cefepime (FEP), ceftazidime (CAZ), ceftriaxone (CRO) and cefotaxime (CTX) of 7.38%, 9.23%, 15.87% and 16.24%, respectively, and resistance rates to levofloxacin (LEV) and ciprofloxacin (CIP) of 8.49% and 19.19%, respectively. It is worth noting that the resistance rates of CRO and CTX in children reached 30.65%. A total of 34 strains carried extended-spectrum β-lactamase (ESBL) genes, dominated by blaCTX-M-65 (13/34) and blaCTX-M-55 (12/34); it is notable that one strain of S. Saintpaul carried both blaCTX-M-27 and blaCTX-M-55. The resistance mechanism to cephalosporins was mainly due to ESBL genes (20/43), and other genes included AmpC and β-lactamase genes. The strains resistant to quinolones mainly carried qnrS1 (27/53), and others included qnrB6, aac(6')-Ib-cr, and mutations in gyrA and parC. One strain did not carry common quinolone resistance genes but had a parC (p.T57S) mutation to cause CIP resistance. This research provides vital insights into the molecular epidemiology and resistance mechanisms of clinical S. enterica, implicating possible infection control strategies.

Genomic characterization of third-generation cephalosporin-resistant Escherichia coli strains isolated from diseased dogs and cats: Report from Japanese Veterinary Antimicrobial Resistance Monitoring

This study investigates the genomic characteristics of canine and feline cefotaxime (CTX, a third-generation cephalosporin)-resistant Escherichia coli using the JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring System, a nationwide monitoring.In this study, whole-genome sequencing (WGS) was performed on 51 canine and 45 feline CTX-resistant E. coli isolates, with certain isolates subjected to pulsed-field gel electrophoresis with S1 nuclease for plasmid–chromosome separation.The most common blaCTX-M genes were blaCTX-M-27 (dogs: 11/51 [21.6 %]; cat: 10/45 [22.2 %]), followed by blaCTX-M-14 (dogs: 10/51 [19.6 %]; cats: 10/45 [22.2 %]), and blaCTX-M-15 (dogs: 9/51 [17.6 %]; cats: 5/45 [11.1 %]). Besides β-lactamase genes, all isolates harbored mdf(A), a multidrug efflux pump, with resistance genes for aminoglycosides, sulfonamides, trimethoprims, macrolides and tetracyclines. None of the isolates had carbapenemase genes, such as blaOXA-48, blaNDM, and blaIMP, whereas most of the isolates showed double mutations in gyrA and parC, which affected quinolone resistance. For the isolates separately analyzed for plasmid and chromosomal DNA via WGS, the majority of CTX-M genes were present on the plasmids. Some plasmids also harbored the same combination of resistance genes and plasmid replicon type, although they differed from isolates derived from different areas of Japan. The predominant plasmids were blaCTX-M-27,aadA5, aph(6)-Id, aph(3”)-Ib, sul1, sul2, tet(A), dfrA17, and mph(A) on IncF. The predominant combination of ST131, O25:H4, and B2 isolates comprised the largest cluster in the minimum spanning tree and the ST131 E. coli harboring blaCTX-M-27 from human in Japan was closely related to these isolates.The results indicated that CTX-resistant canine and feline E. coli harbored multiple plasmids carrying the same combination of resistance genes and emphasizes the need to prevent the spread. Data AvailabilityAll raw short-read sequence data have been deposited in the DNA Data Bank of Japan. (DRR Run No, DRR335726–335821).

Virulent shiga toxin-producing Escherichia coli (STEC) O157:H7 ST11 isolated from ground beef in Brazil.

In this study, a total of 248 ground beef samples were analyzed for the presence of Shiga toxin-producing Escherichia coli (STEC). Out of these samples, only one (0.4%) tested positive for STEC. Further analysis using PCR confirmed the presence of all tested genes associated with STEC, including stx1, stx2, eae, ehx, uid, rfbO157, and fliCH7 in this isolate. Interestingly, no STEC strains were detected in the remaining 100 beef cut samples or the 100 chicken cut samples, indicating the absence of detectable STEC contamination in those specific samples. The isolated strain exhibited significant cytotoxic activity in Vero cells, indicating its ability to produce cytotoxic Shiga toxins. To further investigate the strain, whole-genome sequencing (WGS) analyses were performed. The resistome analysis revealed the absence of acquired antimicrobial resistance genes, indicating a pan-susceptible phenotype. However, this strain presented chromosomal mutations in gyrA, gyrB, parC, parE, pmrA, pmrB, and folP. Plasmid analysis identified the presence of two plasmids, namely IncFIB(AP001918) and IncFII. The multi-locus sequence typing (MLST) identified the strain as belonging to sequence type (ST) 11, which is associated with E. coli O157:H7 strains. The virulome analysis confirmed the presence of several canonical virulence markers, including stx1, stx2, eae-g01-gamma, ehxA, stx1a-O157, and stx2a-O157. Overall, this study identified for the first time a rare occurrence of STEC contamination in ground beef, with the isolated strain belonging to the highly virulent O157:H7 serotype. These findings contribute to our understanding of STEC prevalence and characteristics in food samples, highlighting the importance of effective food safety measures to prevent potential health risks associated with STEC contamination.

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.

Role of parC Mutations at Position 84 on High-Level Delafloxacin Resistance in Methicillin-Resistant Staphylococcus aureus.

High-level delafloxacin-resistant (H-L DLX-R) Staphylococcus aureus isolates (minimum inhibitory concentration ≥1 mg/L) associated with mutations affecting position 84 of ParC have emerged. We aimed to elucidate the role of these mutations as a mechanism of H-L DLX resistance in methicillin-resistant S. aureus (MRSA) isolates recovered from blood cultures. Susceptibility to DLX was determined in 75 MRSA isolates by E-test, and an rt-PCR was developed to detect mutations affecting position 84 of ParC to screen a further 185 MRSA isolates. The genomes of 48 isolates, including all DLX-R isolates or with alterations at position 84, and also a subset of DLX-susceptible isolates were analyzed. Among the 75 isolates studied, 77.34% were DLX-susceptible and only 4 H-L DLX-R isolates were found. Seven (3.8%) isolates with alterations at position 84 of ParC were detected by rt-PCR. Genomic analysis showed that 89.9% (8/9) of isolates with the substitution E84K/G in ParC, together with other mutations in gyrA and parC, were H-L DLX-R. However, the E84K substitution in ParC alone or with other alterations was found in two isolates without H-L DLX-R. Alterations at position 84 of ParC are rare but play a key role in H-L DLX resistance in MRSA but only when other alterations in GyrA are present.

Serovar and sequence type distribution and phenotypic and genotypic antimicrobial resistance of Salmonella originating from pet animals in Chongqing, China.

A total of 334 Salmonella isolates were recovered from 6,223 pet rectal samples collected at 50 pet clinics, 42 pet shops, 7 residential areas, and 4 plazas. Forty serovars were identified that included all strains except for one isolate that did not cluster via self-agglutination, with Salmonella Typhimurium monophasic variant, Salmonella Kentucky, Salmonella Enteritidis, Salmonella Pomona, and Salmonella Give being the predominant serovars. Fifty-one sequence types were identified among the isolates, and ST198, ST11, ST19, ST451, ST34, and ST155 were the most common. The top four dominant antimicrobials to which isolates were resistant were sulfisoxazole, ampicillin, doxycycline, and tetracycline, and 217 isolates exhibited multidrug resistance. The prevalence of β-lactamase genes in Salmonella isolates was 59.6%, and among these isolates, 185 harbored blaTEM, followed by blaCTX-M (66) and blaOXA (10). Moreover, six PMQR genes, namely, including qnrA (4.8%), qnrB (4.2%), qnrD (0.9%), qnrS (18.9%), aac(6')-Ib-cr (16.5%), and oqxB (1.5%), were detected. QRDR mutations (76.6%) were very common in Salmonella isolates, with the most frequent mutation in parC (T57S) (47.3%). Furthermore, we detected six tetracycline resistance genes in 176 isolates, namely, tet(A) (39.5%), tet(B) (8.1%), tet(M) (7.7%), tet(D) (5.4%), tet(J) (3.3%), and tet(C) (1.8%), and three sulfonamide resistance genes in 303 isolates, namely, sul1 (84.4%), sul2 (31.1%), and sul3 (4.2%). Finally, we found 86 isolates simultaneously harboring four types of resistance genes that cotransferred 2-7 resistance genes to recipient bacteria. The frequent occurrence of antimicrobial resistance, particularly in dogs and cats, suggests that antibiotic misuse may be driving multidrug-resistant Salmonella among pets.IMPORTANCEPet-associated human salmonellosis has been reported for many years, and antimicrobial resistance in pet-associated Salmonella has become a serious public health problem and has attracted increasing attention. There are no reports of Salmonella from pets and their antimicrobial resistance in Chongqing, China. In this study, we investigated the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella strains isolated from pet fecal samples in Chongqing. In addition, β-lactamase, QRDR, PMQR, tetracycline and sulfonamide resistance genes, and mutations in QRDRs in Salmonella isolates were examined. Our findings demonstrated the diversity of serovars and sequence types of Salmonella isolates. The isolates were widely resistant to antimicrobials, notably with a high proportion of multidrug-resistant strains, which highlights the potential direct or indirect transmission of multidrug-resistant Salmonella from pets to humans. Furthermore, resistance genes were widely prevalent in the isolates, and most of the resistance genes were spread horizontally between strains.

Prevalence, serotype, antimicrobial susceptibility, contamination factors, and control methods of Salmonella spp. in retail fresh fruits and vegetables: A systematic review and meta-analysis.

This research presents a comprehensive review of Salmonella presence in retail fresh fruits and vegetables from 2010 to 2023, utilizing data from recognized sources such as PubMed, Scopus, and Web of Science. The study incorporates a meta-analysis of prevalence, serovar distribution, antimicrobial susceptibility, and antimicrobial resistance genes (ARGs). Additionally, it scrutinizes the heterogeneous sources across various food categories and geographical regions The findings show a pooled prevalence of 2.90% (95% CI: 0.0180-0.0430), with an increase from 4.63% in 2010 to 5.32% in 2022. Dominant serovars include S. Typhimurium (29.14%, 95% CI: 0.0202-0.6571) and S. Enteritidis (21.06%, 95% CI: 0.0181-0.4872). High resistance rates were noted for antimicrobials like erythromycin (60.70%, 95% CI: 0.0000-1.0000) and amoxicillin (39.92%, 95% CI: 0.0589-0.8020). The most prevalent ARGs were blaTEM (80.23%, 95% CI: 0.5736-0.9692) and parC mutation (66.67%, 95% CI: 0.3213-0.9429). Factors such as pH, water activity, and nutrient content, along with external factors like the quality of irrigation water and prevailing climatic conditions, have significant implications on Salmonella contamination. Nonthermal sterilization technologies, encompassing chlorine dioxide, ozone, and ultraviolet light, are emphasized as efficacious measures to control Salmonella. This review stresses the imperative need to bolster prevention strategies and control measures against Salmonella in retail fresh fruits and vegetables to alleviate related food safety risks.

In vitro induction and selection of fluoroquinolone-resistant mutants in Elizabethkingia anophelis

For 29 parent strains, recognized by pulsed-field gel electrophoresis, the MICs multiplied significantly in the ciprofloxacin group than levofloxacin group, following the first and third induction cycle. Ser83Arg in GyrA was the most common site of mutations. No mutation in ParC nor ParE was identified in the selected mutants.

Whole genome sequencing of uropathogenic E. coli from Ireland reveals diverse resistance mechanisms and strong correlation with phenotypic (EUCAST) susceptibility testing

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes.In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets.Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6′)-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB.These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.

Genetic determinants of antimicrobial resistance in polymyxin B resistant Pseudomonas aeruginosa isolated from airways of patients with cystic fibrosis.

Pseudomonas aeruginosa is the main pathogen associated with pulmonary exacerbation in patients with cystic fibrosis (CF). CF is a multisystemic genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator gene, which mainly affects pulmonary function. P. aeruginosa isolated from individuals with CF in Brazil is not commonly associated with multidrug resistance (MDR), especially when compared to global occurrence, where the presence of epidemic clones, capable of expressing resistance to several drugs, is often reported. Due to the recent observations of MDR isolates of P. aeruginosa in our centers, combined with these characteristics, whole-genome sequencing was employed for analyses related to antimicrobial resistance, plasmid identification, search for phages, and characterization of CF clones. All isolates in this study were polymyxin B resistant, exhibiting diverse mutations and reduced susceptibility to carbapenems. Alterations in mexZ can result in the overexpression of the MexXY efflux pump. Mutations in oprD, pmrB, parS, gyrA and parC may confer reduced susceptibility to antimicrobials by affecting permeability, as observed in phenotypic tests. The phage findings led to the assumption of horizontal genetic transfer, implicating dissemination between P. aeruginosa isolates. New sequence types were described, and none of the isolates showed an association with epidemic CF clones. Analysis of the genetic context of P. aeruginosa resistance to polymyxin B allowed us to understand the different mechanisms of resistance to antimicrobials, in addition to subsidizing the understanding of possible relationships with epidemic strains that circulate among individuals with CF observed in other countries.

Changing Landscape of Antimicrobial Resistance in Neonatal Sepsis: An in silico Analyses of Multidrug Resistance in Klebsiella pneumoniae.

Neonatal sepsis poses a critical healthcare concern, as multidrug-resistant Klebsiella pneumoniae ( K. pneumoniae ) infections are on the rise. Understanding the antimicrobial susceptibility patterns and underlying resistance mechanism is crucial for effective treatment. This study aimed to comprehensively investigate the antimicrobial susceptibility patterns of K. pneumoniae strains responsible for neonatal sepsis using in silico tools. We sought to identify trends and explore reasons for varying resistance levels, particularly for β-lactams and fluoroquinolone. K. pneumoniae isolated from neonates at Kanchi Kamakoti CHILDS Trust Hospital (2017-2020) were analyzed for antimicrobial resistance. Elevated resistance to β-lactam and fluoroquinolone antibiotics was further investigated through molecular docking and interaction analysis. β-lactam affinity with penicillin-binding proteins and β-lactamases was examined. Mutations in ParC and GyrA responsible for quinolone resistance were introduced to investigate ciprofloxacin interactions. Of 111 K. pneumoniae blood sepsis isolates in neonates, high resistance was detected to β-lactams such as cefixime (85.91%, n = 71), ceftriaxone (84.9%, n = 106), cefotaxime (84.9%, n = 82) and fluoroquinolone (ciprofloxacin- 79.44%, n = 107). Molecular docking revealed low β-lactam binding toward penicillin-binding proteins and higher affinities for β-lactamases, attributing to the reduced β-lactam efficiency. Additionally, ciprofloxacin showed decreased affinity toward mutant ParC and GyrA in comparison to their corresponding wild-type proteins. Our study elucidates altered resistance profiles in neonatal sepsis caused by K. pneumoniae , highlighting mechanisms of β-lactam and fluoroquinolone resistance. It underscores the urgent need for the development of sustainable therapeutic alternatives to address the rising antimicrobial resistance in neonatal sepsis.

Development of an in vitro biofilm model for the study of the impact of fluoroquinolones on sewer biofilm microbiota.

Sewer biofilms are likely to constitute hotspots for selecting and accumulating antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to optimize culture conditions to obtain in vitro biofilms, mimicking the biofilm collected in sewers, to study the impact of fluoroquinolones (FQs) on sewer biofilm microbiota. Biofilms were grown on coupons in CDC Biofilm Reactors®, continuously fed with nutrients and inoculum (1/100 diluted wastewater). Different culture conditions were tested: (i) initial inoculum: diluted wastewater with or without sewer biofilm, (ii) coupon material: concrete vs. polycarbonate, and (iii) time of culture: 7 versus 14 days. This study found that the biomass was highest when in vitro biofilms were formed on concrete coupons. The biofilm taxonomic diversity was not affected by adding sewer biofilm to the initial inoculum nor by the coupon material. Pseudomonadales, Burkholderiales and Enterobacterales dominated in the sewer biofilm composition, whereas in vitro biofilms were mainly composed of Enterobacterales. The relative abundance of qnrA, B, D and S genes was higher in in vitro biofilms than sewer biofilm. The resistome of sewer biofilm showed the highest Shannon diversity index compared to wastewater and in vitro biofilms. A PCoA analysis showed differentiation of samples according to the nature of the sample, and a Procrustes analysis showed that the ARG changes observed were linked to changes in the microbial community. The following growing conditions were selected for in vitro biofilms: concrete coupons, initial inoculation with sewer biofilm, and a culture duration of 14 days. Then, biofilms were established under high and low concentrations of FQs to validate our in vitro biofilm model. Fluoroquinolone exposure had no significant impact on the abundance of qnr genes, but high concentration exposure increased the proportion of mutations in gyrA (codons S83L and D87N) and parC (codon S80I). In conclusion, this study allowed the determination of the culture conditions to develop an in vitro model of sewer biofilm; and was successfully used to investigate the impact of FQs on sewer microbiota. In the future, this setup could be used to clarify the role of sewer biofilms in disseminating resistance to FQs in the environment.

Prevalence and molecular characterization of multi-resistant Escherichia coli isolates from clinical bovine mastitis in China

Different antibiotics are used to treat mastitis in dairy cows that is caused by Escherichia coli (E. coli). Antimicrobial resistance in food-producing animals in China has been monitored since 2000. Surveillance data have shown that the prevalence of multiresistant E. coli in animals has increased significantly. This study aimed to investigate the occurrence and molecular characteristics of resistance determinants in E. coli strains (n = 105) obtained from lactating cows with clinical bovine mastitis (CBM) in China. A total of 220 cows with clinical mastitis, which has swollen mammary udder with reduced and red or gangrenous milk, were selected from 5000 cows. The results showed 94.3% of the isolates were recognized as multidrug resistant. The isolates (30.5%) were positive for the class I integrase gene along with seven gene cassettes that were accountable for resistance to trimethoprim resistance (dfrA17, dfr2d and dfrA1), aminoglycosides resistance (aadA1 and aadA5) and chloramphenicol resistance (catB3 and catB2), respectively. The bla TEM gene was present in all the isolates, and these carried the bla CTX gene. A double mutation in gyrA (i.e., Ser83Leu and Asp87Asn) was observed in all fluoroquinolone-resistant isolates. In total, nine fluoroquinolone-resistant E. coli isolates were identified with five different types of mutations in parC. In four (44.4%) isolates, Ser458Ala was present in parE, and in all nine (9/9) fluoroquinolone-resistant isolates, Pro385Ala was present in gyrB. Meanwhile, fluoroquinolone was observed as highly resistant, especially in isolates with gyrA and parC mutations. In summary, the findings of this research recognize the fluoroquinolone resistance mechanism and disclose integron prevalence and ESBLs in E. coli isolates from lactating cattle with CBM.

Occurrence and molecular characteristics of antimicrobial resistance, virulence factors, and extended-spectrum β-lactamase (ESBL) producing Salmonella enterica and Escherichia coli isolated from the retail produce commodities in Bangkok, Thailand

The incidence of antimicrobial resistance (AMR) in the environment is often overlooked and leads to serious health threats under the One Health paradigm. Infection with extended-spectrum β-lactamase (ESBL) producing bacteria in humans and animals has been widely examined, with the mode of transmission routes such as food, water, and contact with a contaminated environment. The purpose of this study was to determine the occurrence and molecular characteristics of resistant Salmonella enterica (S. enterica) (n = 59) and Escherichia coli (E. coli) (n = 392) isolated from produce commodities collected from fresh markets and supermarkets in Bangkok, Thailand. In this study, the S. enterica isolates exhibited the highest prevalence of resistance to tetracycline (11.9%) and streptomycin (8.5%), while the E. coli isolates were predominantly resistant to tetracycline (22.5%), ampicillin (21.4%), and sulfamethoxazole (11.5%). Among isolates of S. enterica (6.8%) and E. coli (15.3%) were determined as multidrug resistant (MDR). The prevalence of ESBL-producing isolates was 5.1% and 1.0% in S. enterica and E. coli, respectively. A minority of S. enterica isolates, where a single isolate exclusively carried blaCTX-M-55 (n = 1), and another isolate harbored both blaCTX-M-55 and blaTEM-1 (n = 1); similarly, a minority of E. coli isolates contained blaCTX-M-55 (n = 2) and blaCTX-M-15 (n = 1). QnrS (11.9%) and blaTEM (20.2%) were the most common resistant genes found in S. enterica and E. coli, respectively. Nine isolates resistant to ciprofloxacin contained point mutations in gyrA and parC. In addition, the odds of resistance to tetracycline among isolates of S. enterica were positively associated with the co-occurrence of ampicillin resistance and the presence of tetB (P = 0.001), while the E. coli isolates were positively associated with ampicillin resistance, streptomycin resistance, and the presence of tetA (P < 0.0001) in this study. In summary, these findings demonstrate that fresh vegetables and fruits, such as cucumbers and tomatoes, can serve as an important source of foodborne AMR S. enterica and E. coli in the greater Bangkok area, especially given the popularity of these fresh commodities in Thai cuisine.

Anthropogenic contamination sources drive differences in antimicrobial-resistant Escherichia coli in three urban lakes.

Antimicrobial resistance (AMR) is an ever-present threat to the treatment of infectious diseases. However, the potential relevance of this phenomenon in environmental reservoirs still raises many questions. Detection of antimicrobial-resistant bacteria in the environment is a critical aspect for understanding the prevalence of resistance outside of clinical settings, as detection in the environment indicates that resistance is likely already widespread. We isolated antimicrobial-resistant Escherichia coli from three urban waterbodies over a 15-month time series, determined their antimicrobial susceptibilities, investigated their population structure, and identified genetic determinants of resistance. We found that E. coli populations at each site were composed of different dominant phylotypes and showed distinct patterns of antimicrobial and multidrug resistance, despite close geographic proximity. Many strains that were genome-sequenced belonged to sequence types of international concern, particularly the ST131 clonal complex. We found widespread resistance to clinically important antimicrobials such as amoxicillin, cefotaxime, and ciprofloxacin, but found that all strains were susceptible to amikacin and the last-line antimicrobials meropenem and fosfomycin. Resistance was most often due to acquirable antimicrobial resistance genes, while chromosomal mutations in gyrA, parC, and parE conferred resistance to quinolones. Whole-genome analysis of a subset of strains further revealed the diversity of the population of E. coli present, with a wide array of AMR and virulence genes identified, many of which were present on the chromosome, including blaCTX-M. Finally, we determined that environmental persistence, transmission between sites, most likely mediated by wild birds, and transfer of mobile genetic elements likely contributed significantly to the patterns observed.IMPORTANCEA One Health perspective is crucial to understand the extent of antimicrobial resistance (AMR) globally, and investigation of AMR in the environment has been increasing in recent years. However, most studies have focused on waterways that are directly polluted by sewage, industrial manufacturing, or agricultural activities. Therefore, there remains a lack of knowledge about more natural, less overtly impacted environments. Through phenotypic and genotypic investigation of AMR in Escherichia coli, this study adds to our understanding of the extent and patterns of resistance in these types of environments, including over a time series, and showed that complex biotic and abiotic factors contribute to the patterns observed. Our study further emphasizes the importance of incorporating the surveillance of microbes in freshwater environments in order to better comprehend potential risks for both human and animal health and how the environment may serve as a sentinel for potential future clinical infections.

Genomic Features and Phylogenetic Analysis of Antimicrobial-Resistant Salmonella Mbandaka ST413 Strains.

In recent years, Salmonella enterica subsp. enterica serovar Mbandaka (S. Mbandaka) has been increasingly isolated from laying hens and shell eggs around the world. Moreover, this serovar has been identified as the causative agent of several salmonellosis outbreaks in humans. Surprisingly, little is known about the characteristics of this emerging serovar, and therefore, we investigated antimicrobial resistance, virulence, and prophage genes of six selected Brazilian strains of Salmonella Mbandaka using Whole Genome Sequencing (WGS). Multi-locus sequence typing revealed that the tested strains belong to Sequence Type 413 (ST413), which has been linked to recent multi-country salmonellosis outbreaks in Europe. A total of nine resistance genes were detected, and the most frequent ones were aac(6')-Iaa, sul1, qacE, blaOXA-129, tet(B), and aadA1. A point mutation in ParC at the 57th position (threonine → serine) associated with quinolone resistance was present in all investigated genomes. A 112,960 bp IncHI2A plasmid was mapped in 4/6 strains. This plasmid harboured tetracycline (tetACDR) and mercury (mer) resistance genes, genes contributing to conjugative transfer, and genes involved in plasmid maintenance. Most strains (four/six) carried Salmonella genomic island 1 (SGI1). All S. Mbandaka genomes carried seven pathogenicity islands (SPIs) involved in intracellular survival and virulence: SPIs 1-5, 9, and C63PI. The virulence genes csgC, fimY, tcfA, sscA, (two/six), and ssaS (one/six) were absent in some of the genomes; conversely, fimA, prgH, and mgtC were present in all of them. Five Salmonella bacteriophage sequences (with homology to Escherichia phage phiV10, Enterobacteria phage Fels-2, Enterobacteria phage HK542, Enterobacteria phage ST64T, Salmonella phage SW9) were identified, with protein counts between 31 and 54, genome lengths of 24.7 bp and 47.7 bp, and average GC content of 51.25%. In the phylogenetic analysis, the genomes of strains isolated from poultry in Brazil clustered into well-supported clades with a heterogeneous distribution, primarily associated with strains isolated from humans and food. The phylogenetic relationship of Brazilian S. Mbandaka suggests the presence of strains with high epidemiological significance and the potential to be linked to foodborne outbreaks. Overall, our results show that isolated strains of S. Mbandaka are multidrug-resistant and encode a rather conserved virulence machinery, which is an epidemiological hallmark of Salmonella strains that have successfully disseminated both regionally and globally.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

2662. Efficacy of Sitafloxacin and Doxycycline Combination Therapy as a Salvage Treatment for Refractory Mycoplasma genitalium Infection

Abstract Background The prevalence of resistant strains of Mycoplasma genitalium has increased over the past few decades, leading to limited treatment options. This study aimed to evaluate the efficacy of sitafloxacin and doxycycline combination therapy as a salvage treatment for refractory rectal and urogenital M. genitalium infections. Methods Men who have sex with men (MSM) diagnosed to have M. genitalium infection from urine samples or rectal swabs using a PCR assay were evaluated and administered sitafloxacin 200 mg and doxycycline 200 mg daily for 21 days. Each sample was tested for 23rRNA, parC, and gyrA mutations before and if a failure after the treatment. Results A total of 27 MSMs were included. The median age was 33 years (range 21-47), and 33.3% (9/27) were people with HIV(PWH) (Table). Among PWH, the median CD4 count was 642, and all were virally suppressed with antiretroviral therapy. Positive samples included 22 rectal and 5 urine specimens. The time to test of cure(TOC) was 14 days (range 0-227). In the two failure cases 9 and 20, the TOC was conducted too early, and it was rechecked after 21 days following treatment completion; both rechecked TOCs were positive. ParC mutations were detected in 100%(23/23) of the samples, gyrA mutations in 66.7%(16/24) and ,macrolide-resistance associated mutations in 100%(22/22). Among the parC mutations, 95.7%(22/23) was G248T(S83I), while 4.3%(1/23) was G259T(D87Y). The overall cure rate was 81.5%. The cure for rectal infection was 81.8%(18/22), while the cure rate for urogenital infection was 80%(4/5). No significant difference was observed between the anatomical sites (p=0.924). The cure rate for strains harboring parC mutation and wild-type in gyrA was 85.7% (6/7), whereas the cure rate for the strain harboring parC mutation and gyrA mutation was 73.3%(11/15). No significant difference was found regardless of gyrA mutations (p=0.519). Additionally, no significant difference was observed in the cure rate between TOC &amp;lt; 21 days and &amp;gt;= 21 days (p=0.964). Cases of Mycoplasma genitalium treatment and antimicrobial-resistance profiles in participants before and after treatment with the combination therapy Conclusion The combination therapy showed high efficacy of 81.5% for highly resistant M. genitalium infection and can be considered as a salvage treatment option for refractory cases. Disclosures All Authors: No reported disclosures