Levofloxacin-resistant Strains Research Articles

Delafloxacin, an alternative treatment for levofloxacin-resistant staphylococci in osteoarticular infections: To use or not to use?

Susceptibility of delafloxacin on 199 osteoarticular levofloxacin-resistant staphylococci strains was reported in 49% and 1% using SSTI S. aureus breakpoint (0.25 mg/L) and general S. aureus breakpoint (0.016 mg/L) respectively. Fifty percents levofloxacin-resistant staphylococci showed resistance to delafloxacin using CA-SFM/EUCAST recommendations. Microbiological societies should define new breakpoints especially for CoNS.

In vitro activity of delafloxacin against clinical levofloxacin-resistant Helicobacter pylori isolates.

Increasing antibiotic resistance in Helicobacter pylori necessitates research on new active molecules. In 2017, delafloxacin, a new fluoroquinolone with chemical properties of activity under acidic conditions, was approved for treatment of community-acquired bacterial pneumonia and acute bacterial skin and soft-tissue infections. Mutations in gyrA are responsible for fluoroquinolone resistance, but certain clinical isolates of H. pylori appear to display a dual phenotype: resistance to levofloxacin associated with very low delafloxacin MICs. To estimate epidemiological cut-off (ECOFF) values and to identify mutations in the gyrA gene, specific to FQ resistance, without increasing the MICs of delafloxacin. Clinical strains (n = 231) were collected in the bacteriology laboratory of Poitiers University Hospital over a 2 year period to determine the ECOFF of delafloxacin. Retrospectively, 101 clinical strains with an levofloxacin-resistant phenotype (MIC > 1 mg/L) were selected from 2018 to 2022 for delafloxacin MIC determination and QRDR (gyrA) sequencing. The estimated ECOFF of delafloxacin was ≤0.125 mg/L. No H. pylori isolate showed a levofloxacin-sensitive phenotype with a delafloxacin MIC of >0.125 mg/L. Among the levofloxacin-resistant H. pylori isolates, 53.5% had delafloxacin MICs of ≤0.125 mg/L. The N87I mutation was associated with dual levofloxacin/delafloxacin resistance (P < 0.001) in contrast to the N87K and D91N mutations (P > 0.05). Mutations D91G and D91Y were not associated with a delafloxacin resistance phenotype (P > 0.05). Delafloxacin seems to be a therapeutic alternative for levofloxacin-resistant strains with greater in vitro activity. However, further clinical/biological investigations are required to determine its efficacy in H. pylori eradication.

Based on molecular docking and real-time PCR technology, the two-component system Bae SR was investigated on the mechanism of drug resistance in CRAB.

This study aimed to explore the role of the two-component system Bae SR in the mechanism of drug resistance in carbapenem-resistant A. baumannii (CRAB) using molecular docking and real-time polymerase chain reaction (PCR). The two-component system Bae SR of Acinetobacter baumannii was subjected to molecular docking with imipenem, meropenem, and levofloxacin. Antibacterial assays and fluorescence quantitative PCR were used to explore protein-ligand interactions and molecular biological resistance mechanisms related to CRAB. The analysis of the two-component system in A. baumannii revealed that imipenem exhibited the highest docking energy in Bae S at - 5.81kcal/mol, while the docking energy for meropenem was - 4.92kcal/mol. For Bae R, imipenem had a maximum docking energy of - 4.28kcal/mol, compared with - 4.60kcal/mol for meropenem. The highest binding energies for Bae S-levofloxacin and Bae R-levofloxacin were - 3.60 and - 3.65kcal/mol, respectively. All imipenem-resistant strains had minimum inhibitory concentration (MIC) values of 16µg/mL, whereas levofloxacin-resistant strains had MIC values of 8µg/mL. The time-sterilization curve showed a significant decrease in bacterial colony numbers at 2h under the action of 8µg/mL imipenem, indicating antibacterial effects. In contrast, levofloxacin did not exhibit any antibacterial activity. Fluorescence quantitative PCR results revealed significantly increased relative expression levels of bae S and bae R genes in the CRAB group, which were 2 and 1.5 times higher than those in the CSAB group, respectively, with statistically significant differences. Molecular docking in this study found that the combination of Bae SR and carbapenem antibiotics (imipenem, meropenem) exhibited stronger affinity and stability compared with levofloxacin. Moreover, the overexpression of the two-component system genes in carbapenem-resistant A. baumannii enhanced its resistance to carbapenem, providing theoretical and practical insights into carbapenem resistance in respiratory tract infections caused by A. baumannii.

Fourth national Japanese antimicrobial susceptibility pattern surveillance program: Bacterial isolates from patients with complicated urinary tract infections

IntroductionAntimicrobial susceptibility patterns of bacterial pathogens isolated from patients with complicated urinary tract infections were analyzed using the national surveillance data, comprising 793 bacterial strains from eight clinically relevant species. Materials and methodsData were collected for the fourth national surveillance project from July 2020 to December 2021 by the Japanese Society of Chemotherapy, the Japanese Association for Infectious Disease, and the Japanese Society of Clinical Microbiology. Surveillance was supervised with the cooperation of 43 medical institutions throughout Japan. ResultsFluoroquinolone required a minimum inhibitory concentration (MIC) of 2–64 mg/L to inhibit the 330 tested Escherichia coli strains. The proportion of levofloxacin-resistant E. coli strains increased from 28.6% in 2008 to 29.6% in 2011, 38.5% in 2015, and 44.5% in 2021. The proportion of levofloxacin-resistant strains of Pseudomonas aeruginosa also increased from previous survey results, showing a continuing downward trend. Conversely, the proportion of levofloxacin-resistant strains of Enterococcus faecalis decreased relative to previous reports. Neither multidrug-resistant P. aeruginosa nor carbapenem-resistant Enterobacteriaceae were detected. For methicillin-resistant Staphylococcus aureus (MRSA), the proportion of vancomycin-susceptible strains (MIC of 2 μg/mL) decreased from 14.7% to 7.7%. DiscussionBacterial strains that produced extended-spectrum β-lactamase included E. coli (82/330 strains, 24.8%), Klebsiella pneumoniae (11/68 strains, 16.2%), and Proteus mirabilis (4/26 strains, 15.4%). As compared to previous surveillance reports, these strains showed an increase in proportion over the years.

Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains.

Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.

The Formation and Drug Resistance Mechanism of Biofilm for Streptococcus pneumoniae Infection in Severe Respiratory Patients.

This study was to explore whether Streptococcus pneumoniae would form biofilms and the formative factors of biofilms, as well as the drug resistance mechanism of S. pneumoniae. In this study, a total of 150 strains of S. pneumoniae were collected from 5 local hospitals in the past two years, and the minimum inhibitory concentrations (MIC) of levofloxacin, moxifloxacin and penicillin were determined by agar double dilution method to select the drug-resistant strains. The polymerase chain reaction (PCR) amplification and sequencing were performed on specific genes of drug-resistant strains. In addition, 5 strains of S. pneumoniae with penicillin MIC ≤ 0.065 μg/mL, 0.5 μg/mL, 2 μg/mL, ≥ 4μg/mL were randomly selected, and the biofilms were cultured on two kinds of well plates for 24 hours. Finally, whether the biofilms were formed was observed. Experimental results revealed that the resistance rate of S. pneumoniae to erythromycin in this area was as high as 90.3%, and the strains that were resistant to penicillin account for only 1.5%. The amplification and sequencing experiment revealed that one (strain 1) of the strains, which was resistant to both drugs, had a GyrA mutation and ParE mutation, and strain 2 had a parC mutation. All strains generated biofilms, and the optical density (OD) value of penicillin MIC ≤ 0.065 μg/mL group (0.235 ± 0.053) was higher than that of 0.5 μg/mL group (0.192 ± 0.073) (P< 0.05) and higher than the OD value of the 4 μg/mL group (0.200 ± 0.041) (P< 0.05), showing statistically great differences. It was confirmed that the resistance rate of S. pneumoniae to erythromycin remained high, the rate of sensitivity to penicillin was relatively high, and the moxifloxacin and levofloxacin-resistant strains had appeared; S. pneumoniae mainly showed QRDR mutations in gyrA, parE, and parC; and it was confirmed that S. pneumoniae can generate biofilms in vitro.

Update on the second-line treatment of Helicobacter pylori infection: a narrative review.

A standard bismuth quadruple therapy, a fluoroquinolone-containing triple (or quadruple) therapy or a proton pump inhibitor (PPI)-amoxicillin high-dose dual therapy has been recommended as a second-line treatment for Helicobacter pylori infection by the Maastricht VI/Florence Consensus Report. The major shortcoming of levofloxacin-amoxicillin triple therapy is low cure rate for eradicating levofloxacin-resistant strains. With the rising prevalence of levofloxacin-resistant strains, levofloxacin-amoxicillin triple therapy cannot reliably achieve a high eradication rate for second-line treatment of H. pylori infection in most countries now. The present article aims to review current second-line eradication regimens with a per-protocol eradication rate exceeding 85% in most geographic areas. Recently, a novel tetracycline-levofloxacin quadruple therapy consisting of a PPI, bismuth, tetracycline, and levofloxacin for rescue treatment of H. pylori infection has been developed. The new therapy achieved a higher per-protocol eradication rate than levofloxacin-amoxicillin triple treatment in a randomized controlled trial (98% versus 69%). Additionally, the tetracycline-levofloxacin quadruple therapy also exhibits a higher eradication rate than amoxicillin-levofloxacin quadruple therapy. High-dose dual PPI-amoxicillin therapy is another novel second-line treatment for H. pylori infection. The new therapy can achieve an eradication rate of 89% by per-protocol analysis for the second-line treatment in Taiwan. Recently, levofloxacin-based sequential quadruple therapy and potassium-competitive acid blocker have also been applied in the second-line treatment of H. pylori infection. A meta-analysis revealed that a vonoprazan-based regimen has significant superiority over a PPI-based regimen for second-line H. pylori eradication therapy. In conclusion, the eradication rate of levofloxacin-amoxicillin triple therapy is suboptimal in the second-line treatment of H. pylori infection now. Currently, a standard bismuth quadruple therapy (tetracycline-metronidazole quadruple therapy), a tetracycline-levofloxacin quadruple therapy, an amoxicillin-levofloxacin quadruple therapy, a levofloxacin-based sequential quadruple therapy or a high-dose PPI-amoxicillin dual therapy is recommended for the second-line treatment of H. pylori infection.

1832. Epidemiology, risk factors for mortality and resistance associated with Stenotrophomonas maltophilia bacteremia at a tertiary care hospital in Korea over 7 years

Abstract Background Stenotrophomonas maltophilia (SM) is an important emerging nosocomial pathogen. This study aimed to describe the epidemiological, clinical, and microbiological charateristics of SM bacteremia at a tertiary care hospital in Korea over 7 years. Methods A retrospective study was conducted at a 1150-bed tertiary care hospital in Busan, South Korea. Data were collected between January, 2013 and November, 2019. Results A total of 79 bacteremia patients (median age: 61 years; 62% males) were reviewed; 38 (48.1%) were in 2013-2016 and 41 (51.9%) were in 2017-2019. The overall SM bacteremia-related 30-days mortality was 31.6%. Susceptibility to levofloxacin, sulfamethoxazole-trimethoprim (SMX/TMP) and minocycline were 87.3%, 92.4% and 100%, respectively. The most common clinical syndrome was central line-associated bloodstream infection (n=37, 46.8%). The others were intra-abdominal infection (n=18, 22.8%), skin and soft tissue infection (n=5, 6.3%) and pneumonia (n=5, 6.3%). In 50.6%, SM was the only pathogen of bacteremia, whereas in 49.4%, more than one causative organisms were isolated. Acinetobacter baumanii (in 8 cases) was the most frequent co-isolated pathogen, followed by Enterococcus faecium (in 6 cases), Pseudomonas aeruginosa (in 5 cases), Staphylococcus epidermidis (in 5 cases), Enterococcus gallinarum (in 3 cases) and Klebsiella pneumoniae (in 3 cases). Age (OR, 6.60; 95% Cl, 1.59-27.41; P = 0.009), inappropriate antimicrobial treatment (OR, 3.47; 95% Cl, 1.14-10.56; P = 0.029), and levofloxacin-resistant strains (OR, 7.86; 95% Cl, 1.20-51.41; P = 0.031) were independent risk factors for SM bacteremia-related 30-days mortality in multivariate analysis. Conclusion SM bacteremia has a high early mortality rate. In our study, old age, inappropriate antimicrobial treatment and levofloxacin-resistant strains were significant independent risk factors for early mortality in SM bacteremia patients. Early diagnosis and immediate use of appropriate antibiotics are recommended to improve mortality of SM bacteremia. Disclosures All Authors: No reported disclosures.

Evaluation of clarithromycin and levofloxacin resistance of Helicobacter pylori strains isolated from patients at the Hospital for Tropical Diseases, Ho Chi Minh City

Clarithromycin (CLA) and levofloxacin (LFX) have been recommended as the most effective antibiotics for treating Helicobacter pylori infection. However, the increase in H. pylori’s resistance to antibiotics is an alarming and growing challenge. The study aimed to determine the antimicrobial resistance profiles of H. pylori strains isolated from patients at the Hospital for Tropical Diseases and to detect point mutations in 23S ribosomal RNA (23S rRNA) and gyraseA (gyrA) genes. Point mutations in 23S rRNA and gyrA were detected using Sanger sequencing. Antibiotic resistance was tested by the microdilution method. Out of the 45 isolates, 44 (97.8%) were found to be resistant to at least one antibiotic, 38 (84.4%) resistant to metronidazole (MTZ), and all were sensitive to amoxicillin (AMX) and tetracycline (TET). Sixty percent of the isolated strains were resistant to 3-6 antibiotics; among them, multiple drug resistance (MDR) strains presented twenty percent resistance to more than two classes of antibiotics. Eleven strains (24.4%) carried two mutations associated with CLA and LFX but only nine of them were dual resistant to CLA and LFX, and twenty-four (53.3%) carried either CLA or LFX mutation. The point mutations A2143G in the 23S rRNA and N87K (Asn87Lys) in the gyrA were detected in the CLA and LFX resistant strains, respectively. The prevalence of MDR, especially CLA, MTZ, and LFX resistance, in the H. pylori isolates suggests that the use of these antibiotics need to be more considerable and cautious.

Clinical and molecular epidemiology of invasive group B Streptococcus infections in adults in a referral center in Korea.

Invasive group B Streptococcus (GBS) infections are increasing among adults with underlying health conditions; however, clinical manifestations and serotype distribution remain unclear. This study investigated the molecular characteristics and antimicrobial resistance of invasive GBS in Korean adults. GBS isolates from patients with invasive diseases during 2006–2015 were investigated for capsular serotype, multilocus sequence type (ST), antimicrobial susceptibility, and resistance genes. Among the 74 isolates analyzed, the most common serotype was Ib (31.1%), followed by III (21.6%), V (20.3%), Ia (12.2%), and VI (12.2%). Thirteen STs were detected, with ST1, ST10, ST19, and ST23 as the most prevalent. The dominant capsular serotype exhibited by ST1 was V, and those expressed by ST10, ST19, and ST23 were Ib, III, and Ia, respectively. Erythromycin and levofloxacin resistance were observed in 33.8% and 31.1% of the isolates, respectively. ST10-Ib (n = 11/11, 100%) and ST654-Ib (n = 3/3, 100%) were dominant levofloxacin-resistant strains. Serotypes Ib, III, and V were most common among adults, which is inconsistent with recent reports in Korea where III, V, and Ia were predominant in infants. The difference in the serotype distribution between adults and children may be associated with the selective pressure imparted by antibiotics.

Efficacy of bismuth for antibiotic-resistant Helicobacter pylori strains eradication: A systematic review and meta-analysis.

Antibiotic resistance of Helicobacter pylori (H.pylori) is increasing worldwide, and bismuth quadruple therapy has been recommended as a first-line regimen in many areas. This study aimed to investigate whether bismuth would improve the eradication rate (ER) of clarithromycin-/metronidazole-/levofloxacin-resistant H.pylori strains and how much additional efficacy bismuth could achieve. PubMed, EMBASE, Web of Science, and Cochrane Central databases for randomized controlled trials were systematically searched by two independent reviewers until 15 January 2022. Pooled ERs of clarithromycin-/metronidazole-/levofloxacin-resistant H.pylori strains were compared between bismuth-containing and non-bismuth therapies. Pooled risk ratios (RRs) with 95% confidence intervals (CIs) were calculated using a random-effects model. Eight studies enrolling 340 individuals were included. The RRs of pooled ERs compared between bismuth-containing and non-bismuth therapies were 1.83 for clarithromycin-resistant strains (95% CI 1.16-2.89, pooled ER: 76.9% vs. 36.6%, p=.009, I2 =0%), 1.39 for metronidazole-resistant strains (95% CI 1.09-1.78, pooled ER: 86.8% vs. 60.9%, p=.008, I2 =37%), 2.75 for dual clarithromycin/metronidazole-resistant strains (95% CI 1.01-7.52, pooled ER: 76.9% vs. 18.2%, p=.05, I2 =0%), and 1.04 for levofloxacin-resistant strains (95% CI 0.56-1.93, pooled ER: 63.4% vs. 54.3%, p=.90; I2 =60%). Bismuth significantly increased the ERs of clarithromycin-, metronidazole-, and dual-resistant strains by 40%, 26%, and 59%, respectively. Subgroup analysis of treatment duration showed that the significantly higher eradication rate for antibiotic-resistant strains in bismuth-containing therapy than non-bismuth therapy was only observed in 14-day treatment regimens and not in 7-day regimens (p=.02 and .17, respectively). Bismuth was most effective in improving the ERs of dual-resistant H.pylori strains, followed by clarithromycin- and metronidazole-resistant strains. Prolonged treatment duration might effectively improve the efficacy of bismuth in overcoming antibiotic resistance.

Antibiotic resistance of Helicobacter pylori isolated from children in Chongqing, China.

The resistance of Helicobacter pylori (H. pylori) to antibiotics has been increasing worldwide and varies across different geographic areas and times. Limited studies reported the prevalence of antibiotic resistance and its related gene mutations in children in Chongqing, a city located in southwest China. We collected 112 H. pylori strains isolated from gastric biopsies of 156 children at Children's Hospital of Chongqing Medical University and calculated resistance rates of these strains to six antibiotics. The A2143G and A2142G mutations in 23S rRNA gene, which are related to clarithromycin resistance, and Asn87 and Asp91 mutations in gyrA gene, which are related to levofloxacin resistance, were investigated in 102 strains. The resistance rates to clarithromycin, metronidazole, and levofloxacin were 47.3% (53/112), 88.4% (99/112), and 18.8% (21/112), respectively. No resistance to amoxicillin, tetracycline, and furazolidone was observed. Dual and triple resistance percentages were 37.5% (42/112) and 10.7% (12/112), respectively. The detection rate of A2143G mutation in 23S rRNA gene was 83.3% (40/48). The detection rates of mutations of Asn87 and Asp91 in gyrA gene were 52.6% (10/19) and 36.8% (7/19), respectively. Conclusion:The prevalence of H. pylori resistance to clarithromycin, metronidazole, and levofloxacin was high in children in Chongqing, China. The A2143G mutation was detected in most clarithromycin-resistant strains, and Asn87 and Asp91 of gyrA mutation points were common in levofloxacin-resistant strains. In clinical practice, anti-H. pylori therapy should be individualized based on a susceptibility test. What is Known: •The resistance of H. pylori to antibiotics changes with the geographic areas and that in Asia the resistance rate is high. • Mutation plays a vital role in antibiotics resistance of H. pylori. What is New: •High resistance rates to single and multiple antibiotics in children of Chongqing, a city located in southwest China, were observed. • Molecular assays showed good conformance with susceptibility test results to direct antibiotic resistance of H. pylori.

Escherichia coli ST131 isolated from urological patients can acquire plasmid-mediated extended spectrum β-lactamase from other bacteria with high frequency.

To investigate the prevalence of the clonal group Escherichia coli ST131 in urologic patients, and to clarify the mechanisms underlying the high prevalence of the antimicrobial resistant genes in ST131. We used 65 Escherichia coli strains collected from the Department of Urology, Nagasaki University Hospital, between January 2018 and December 2018. All of them underwent multilocus sequence typing and were analyzed for genes associated with quinolone resistance and extended-spectrum β-lactamases. To compare ST131 and non-ST131 strains, bacterial conjugation experiments and intestinal colonization evaluations were performed. ST131 was the most dominant among all the strains, along with levofloxacin resistant strains, and extended-spectrum β-lactamases positive strains (32%, 63%, and 73%, respectively). 12 out of 15 extended-spectrum β-lactamases-producing Escherichia coli strains harbored CTX-M-9. In particular, all extended-spectrum β-lactamases-producing ST131 strains possessed CTX-M-9. The proportions of ST131 strains with or without quinolone resistance-determining region mutations were significantly higher and lower, respectively, than that of non-ST131 strains (P = 0.0002 and P < 0.0001, respectively). When Klebsiella pneumoniae was used as a donor, three ST131 strains acquired extended-spectrum β-lactamases a total of 16 times (six, four, and six times each), which was significantly more than that in one of the non-ST131 strains (two times). The amount of bacteria was significantly lower in the ST131 strains than in the non-ST131 strains administered to mice. Both the ST131 and non-ST131 strains increased again after the administration of vancomycin, even after the colony was not detected. These results support the mechanisms underlying the prevalence of ST131 strains in hospitals, particularly in urologic patients.

Clinical and Microbiological Characterization of Bloodstream Infections Caused by Mycoplasma hominis: An Overlooked Pathogen.

IntroductionBloodstream infection (BSI) is associated with high mortality rates. Mycoplasma hominis, which rarely causes extragenital infections, has been shown to induce BSI and presents a clinical diagnostic and therapeutic challenge.MethodsIn this study, we investigated the clinical characteristics, antibiotic resistance, and multilocus sequence typing (MLST) of eight BSI cases caused by M. hominis in South China from January 2018 to October 2021.ResultsUnderlying immunosuppression and genitourinary tract surgery are important risk factors for M. hominis BSI. The most prevalent clinical symptoms and signs were fever. Additional findings included elevated neutrophil count and C-reactive protein level. Furthermore, in this study, all the patients had erythrocytopenia. M. hominis harbored the highest rate of resistance to levofloxacin (75.0%), followed by sparfloxacin (50.0%), and gatifloxacin (37.5%). gyrA S153L was the most frequent mutation in levofloxacin-resistant strains, followed by parC S91I. parC K144R may be related to resistance to gatifloxacin and sparfloxacin. Eight strains showed sensitivity to all the other antibiotics analyzed (doxycycline, minocycline, josamycin, and clindamycin). MLST was performed in seven isolates, and seven new sequence types were described. We compared our isolates with all M. hominis strains from the PubMLST database, and one major clonal complex and eight singletons were identified.ConclusionsOur study clarified and expanded the clinical features and antibiotic resistance of M. hominis BSI. These findings are useful for the clinical diagnosis and control of M. hominis BSI.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40121-022-00616-w.

Development and Validation of Multiplex Quantitative PCR Assay for Detection of Helicobacter pylori and Mutations Conferring Resistance to Clarithromycin and Levofloxacin in Gastric Biopsy.

Aims and ObjectivesMore than half of the world’s population is infected with Helicobacter pylori, which can cause chronic gastritis. WHO has regarded clarithromycin-resistant H. pylori as a high priority pathogen. Hence, accurate diagnosis and detection of clarithromycin- and levofloxacin-resistant H. pylori strains is essential for proper management of infection. The objective of this study was to develop and optimize multiplex quantitative PCR assay for detection of mutations associated with clarithromycin and levofloxacin resistance in H. pylori directly from the gastric biopsies.Materials and MethodsSpecific primers and probes were designed to amplify ureA and mutations in 23S rRNA and gyrA genes. Singleplex and triplex qPCR assays were optimized and the assay’s sensitivities and specificities were determined. The optimized multiplex qPCR assay was performed on 571 gastric biopsies.ResultsIn this study, 14.7% (84/571) of the gastric biopsies were positive for H. pylori by conventional methods and 23.8% (136/571) were positive by the ureA-qPCR with 96.4% sensitivity and 88.5% specificity, while the +LR and −LR were 8.72 and 0.04, respectively. The ureA-positive samples (n=136) were subjected to multiplex qPCR which detected A2142G and A2143G mutations in the 23S rRNA gene (20.6%, 28/136) conferring clarithromycin resistance and gyrA mutations N87K, N87I, D91N, and D91Y (11.8%, 16/136) leading to levofloxacin resistance. The sensitivity and specificity of qPCR of 23S rRNA gene were 100% and 98.7%, respectively, while 100% and 99.8% for qPCR of gyrA, respectively.ConclusionThe effectiveness of this qPCR is that it is sensitive in detecting low bacterial load and will help in timely detection of clarithromycin- and levofloxacin-resistant strains, especially in case of mixed infections. Since it is culture independent, it can inform clinicians about antibiotics to be included in the first-line therapy, thereby improving the management of H. pylori infection at a much greater pace.

Tetracycline-levofloxacin versus amoxicillin-levofloxacin quadruple therapies in the second-line treatment of Helicobacter pylori infection.

The Maastricht V/Florence Consensus Report recommends amoxicillin-fluoroquinolone triple or quadruple therapy as a second-line treatment for Helicobacter pylori infection. An important caveat of amoxicillin-fluoroquinolone rescue therapy is poor eradication efficacy in the presence of fluoroquinolone resistance. The study aimed to investigate the efficacies of tetracycline-levofloxacin (TL) quadruple therapy and amoxicillin-levofloxacin (AL) quadruple therapy in the second-line treatment of H.pylori infection. Consecutive H. pylori-infected subjects after the failure of first-line therapies were randomly allocated to receive either TL quadruple therapy (tetracycline 500mg QID, levofloxacin 500mg QD, esomeprazole 40mg BID, and tripotassium dicitrato bismuthate 300mg QID) or AL quadruple therapy (amoxicillin 500mg QID, levofloxacin 500mg QD, esomeprazole 40mg BID, and tripotassium dicitrato bismuthate 300mg QID) for 10days. Post-treatment H.pylori status was assessed 6weeks after the end of therapy. The study was early terminated after an interim analysis. In the TL quadruple group, 50 out of 56 patients (89.3%) had successful eradication of H.pylori infection. Cure of H.pylori infection was achieved only in 39 of 52 patients (69.6%) receiving AL quadruple therapy. Intention-to-treat analysis showed that TL quadruple therapy achieved a markedly higher eradication rate than AL quadruple therapy (95% confidence interval: 4.8% to 34.6%; p=0.010). Further analysis revealed that TL quadruple therapy had a high eradication rate for both levofloxacin-susceptible and resistant strains (100% and 88.9%). In contrast, AL quadruple therapy yielded a high eradication for levofloxacin-susceptible strains (90.9%) but a poor eradication efficacy for levofloxacin-resistant strains (50.0%). The two therapies exhibited comparable frequencies of adverse events (37.5% vs 21.4%) and drug adherence (98.2% vs 94.6%). Ten-day TL quadruple therapy is more effective than AL quadruple therapy in the second-line treatment of H. pylori infection in a population with high levofloxacin resistance.

Efficacy and Safety of Antofloxacin-Based Triple Therapy for Helicobacter pylori Eradication Failure in China.

Quinolone-containing triple therapy has been considered as the second-line therapy for eradication of Helicobacter pylori (H. pylori). At present, there are no data to show the efficacy and safety of antofloxacin-based rescue therapy for the eradication of H. pylori, and this pilot clinical trial was designed. A total of 196 patients who failed H. pylori eradication using the clarithromycin-based or metronidazole-based triple or bismuth quadruple therapy were randomly allocated to one of the following rescue eradication therapy groups: AEA group (antofloxacin 200mg once daily, esomeprazole 20mg + amoxicillin 1000mg twice daily) for 14days, or LEA group (levofloxacin 500mg once daily, esomeprazole 20mg + amoxicillin 1000mg twice daily) for 14days. The minimal inhibitory concentrations were tested by the E-test method. The gyrA mutation was analyzed by sequencing. Follow-up 13/14C-urea breath test was examined at 1month after discontinuation. A total of 178 eligible patients were included in this study. The eradication rate was significantly higher in AEA group than in LEA group according to both ITT (87.6% vs. 68.5%; P = 0.002) and PP analyses (90.7% vs. 70.1%; P = 0.001). ITT analyses indicated that the eradication rate was significantly higher in AEA group than in LEA group with Asn87 mutation (78.9% vs. 31.3%; P = 0.005) and levofloxacin-resistant strains (76.9% vs. 44.2%; P = 0.003). Two groups exhibited similar adverse event rates (AEA 14.6% vs. LEA 20.2%, P = 0.323). The findings showed that antofloxacin may be a promising candidate in rescue therapy for H. pylori eradication failure in China.

Point Mutations at gyrA and gyrB Genes of Levofloxacin Resistant Helicobacter pylori Strains and Dual Resistance with Clarithromycin.

Spontaneous point mutations in genes encoding gyrA/B subunits of DNA gyrase are responsible for fluoroquinolone resistance. We aimed to determine the clarithromycin and levofloxacin resistance phenotypically in H. pylori strains and to investigate the mutations responsible for levofloxacin resistance and the effects of these mutations on dual antibiotic resistance. A total of 65 H. pylori isolates were included. The E-test method was used for the clarithromycin and le-vofloxacin antimicrobial susceptibility test. Real-time PCR was used to detect the point mutations. Twenty-four (36.9%) of 65 H. pylori strains were phenotypically resistant to clarithromycin and 14 (21.5%) to levofloxacin. The phenotypic levofloxacin resistance rate of strains with Asn87Lys and Asp91Asn mutations were significantly higher (gyrA gene) (p < 0.05). The phenotypic levofloxacin resistance rate of strains with Arg484Lys and Asp481Glu mutations were significantly higher (gyrB gene) (p < 0.05). The Asn87Lys mutation increased the risk of phenotypes being resistant to levofloxacin 70.156 times and Asp91Asn mutation increased 125,427 times higher. Seven (10.8%) of 65 H. pylori strains showed dual resistance to both levofloxacin and clarithromycin. The rate of being dual resistant with A2143G mutation (clarithromycin resistance) was found to be significantly higher (p < 0.05). The Asn87Lys and Asp91Asn mutations in the gyrA gene had a phenotypically enhancing effect on levofloxacin resistance, while the presence of Asp481Glu and Arg484Lys mutations in the gyrB gene did not. The existence of dual resistance was developed with the increase in clarithromycin and levofloxacin resistance rates.

Adjunctive Use of Phage Sb-1 in Antibiotics Enhances Inhibitory Biofilm Growth Activity versus Rifampin-Resistant Staphylococcus aureus Strains.

Effective antimicrobials are crucial for managing Staphylococcus aureus implant-associated bone infections (IABIs), particularly for infections due to rifampin-resistant S. aureus (RRSA). Failure to remove the implant results in persistent infection; thus, prolonged suppressive antibiotic therapy may be a reasonable alternative. However, a high incidence of adverse events can necessitate the discontinuation of therapy. In this scenario, commercial Staphylococcal bacteriophage Sb-1 combined with antibiotics is an option, showing a promising synergistic activity to facilitate the treatment of biofilm infections. Therefore, we evaluated the efficacy of the inhibitory activity of five antibiotics (doxycycline, levofloxacin, clindamycin, linezolid, and rifampin) alone or combined with phage Sb-1 (106 PFU/mL) in a simultaneous and staggered manner, to combat five clinical RRSA strains and the laboratory strain MRSA ATCC 43300 in 72 h by isothermal microcalorimetry. The synergistic effects were observed when phage Sb-1 (106 PFU/mL) combined with antibiotics had at least 2 log-reduction lower concentrations, represented by a fractional biofilm inhibitory concentration (FBIC) of <0.25. Among the antibiotics that we tested, the synergistic effect of all six strains was achieved in phage/doxycycline and phage/linezolid combinations in a staggered manner, whereas a distinctly noticeable improvement in inhibitory activity was observed in the phage/doxycycline combination with a low concentration of doxycycline. Moreover, phage/levofloxacin and phage/clindamycin combinations also showed a synergistic inhibitory effect against five strains and four strains, respectively. Interestingly, the synergistic inhibitory activity was also observed in the doxycycline-resistant and levofloxacin-resistant profile strains. However, no inhibitory activity was observed for all of the combinations in a simultaneous manner, as well as for the phage/rifampin combination in a staggered manner. These results have implications for alternative, combined, and prolonged suppressive antimicrobial treatment approaches.

Helicobacter pylori Primary and Secondary Genotypic Resistance to Clarithromycin and Levofloxacin Detection in Stools: A 4-Year Scenario in Southern Italy.

Antibiotic resistance has become an emerging problem for treating Helicobacter pylori (H. pylori) infection. Clarithromycin and levofloxacin are two key antibiotics used for its eradication. Therefore, we reviewed our experience with genotypic resistance analysis in stools to both clarithromycin and levofloxacin in the last four years to evaluate time trends, both in naive and failure patients. Patients collected a fecal sample using the THD fecal test device. Real-time polymerase chain reaction was performed to detect point mutations conferring resistance to clarithromycin (A2142C, A2142G, and A2143G in 23S rRNA) and levofloxacin (substitutions at amino acid position 87 and 91 of gyrA). One hundred and thirty-five naive patients were recruited between 2017–2020. Clarithromycin resistance was detected in 37 (27.4%). The time trend did not show any significant variation from 2017 to 2020 (p = 0.33). Primary levofloxacin resistance was found in 26 subjects (19.2%), and we observed a dramatic increase in rates from 2017 (10%) to 2018 (3.3%), 2019 (20%), and 2020 (37.8%). Ninety-one patients with at least one eradication failure were recruited. Secondary resistance to clarithromycin and levofloxacin was found in 59 (64.8%) and 45 patients (59.3%), respectively. In conclusion, our geographic area has a high risk of resistance to clarithromycin. There is also a progressive spreading of levofloxacin-resistant strains.