Fluoroquinolone Resistance Research Articles

First Report of Campylobacter jejuni Strains Belonging to ST-21 Clonal Complex Isolated from Human, Poultry and Wild Birds in Croatia: Antimicrobial Resistance and Genetic Distance.

In the era of growing antimicrobial resistance, a threat affecting humans, endangering animals, as well as livelihoods and food security worldwide, we wanted to find possible explanations for its continuous spread from a new perspective. The ubiquity of resistance genes requires a One Health approach to finding the explanations for continuous AMR spread. The natural transformability of Campylobacter jejuni, its high incidence of infections, and emerging resistance worldwide inspired us to choose C. jejuni ST-21CC to be our pathogen for analyzing its contribution and connection to the cycle of AMR dissemination. ST-21CC is known as a generalist among humans and broilers, the most prevalent lineage worldwide, but it is rarely found in wild birds. Emerging in wild birds, genetic relatedness and similar resistance profiles were expected. We analyzed 23 Croatian C. jejuni strains belonging specifically to ST-21CC from humans, broilers, and wild birds. The genomic data obtained through whole genome sequencing and phenotypic susceptibility data of strains were compared. Our findings suggest high fluoroquinolone resistance in ST-21CC strains, with more diverse genetic backgrounds in wild birds. Intriguing were three isolates of ST-822 (from human and storks), sharing a similar genetic fingerprint.

Doxycycline post exposure prophylaxis could select for cross-resistance to other antimicrobials in various pathogens: An in silico analysis.

A number of randomized controlled trials have found that doxycycline post exposure prophylaxis (PEP) can reduce the incidence of gonorrhoea, chlamydia and syphilis in men who have sex with men (MSM). If tetracycline resistance is associated with resistance to other antimicrobials in a range of bacterial species, then doxycycline PEP could have the unintended effect of selecting for resistance to other antimicrobials in these bacterial species. Antimicrobial susceptibility data were retrieved from two sources: pubMLST (https://pubmlst.org/) and Pathogenwatch (https://pathogen.watch/) for the following bacterial pathogens: Klebsiella pneumoniae, Salmonella enterica subsp. Enterica serovar Typhi, Campylobacter jejuni, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus pyogenes. We assessed if tetracycline resistance was associated with resistance to six relevant antimicrobials. We found evidence of cross resistance to various antimicrobials in all six bacterial species assessed. Cross resistance was found in 4 of 5 antimicrobials for K. pneumoniae, 1 of 2 for C. jejuni, 3 of 5 for S. enterica subsp. Enterica serovar Typhi, 5 of 5 for S. aureus, 5 of 6 for S. pneumoniae and 2 of 3 for S. pyogenes. These associations include a higher prevalence of methicillin resistance in tetracycline resistant S. aureus, penicillin resistance in S. pneumoniae, macrolide and clindamycin resistance in S. pyogenes, fluoroquinolone resistance in S. enterica subsp. Enterica serovar Typhi and third-generation cephalosporin resistance in K. pneumoniae. These results suggest that studies evaluating the effects of doxycycline PEP should include the effects of doxycycline on resistance not only to doxycycline but also to other antimicrobials and in a broader array of bacterial species than has been included in doxycycline PEP studies thus far.

Analysis of Mycobacterium tuberculosis Complex Strains Isolated from the Samples of Patients Living in Northern Syria

Tuberculosis causes serious mortality and morbidity worldwide each year. A lot of effort and money is spent for the diagnosis and treatment of tuberculosis all over the world. The importance that countries give to health policies and public health is inversely proportional to the incidence of tuberculosis and multidrug resistant tuberculosis. The aim of our study was to evaluate the resistance profiles of Mycobacterium tuberculosis complex strains which were isolated from sputum samples, collected by World Health Organisation from patients living in the northern region of Syria, where health services were disrupted due to the civil war. According to the protocol signed between the World Health Organization and our hospital; sputum samples taken from tuberculosis patients living in Afrin, Azez and Idlib regions or suspected of being resistant to anti-tuberculosis drugs were studied in our hospital. The cultivation process was performed in our laboratory using Löwenstein Jensen media and MGIT-960 system. The susceptibility tests for primary anti-tuberculosis drugs were performed using MGIT-960 system for M.tuberculosis complex isolates. The isolates identified as MDR/RD-TB (multi-drug-resistant-rifampicin-resistant tuberculosis) were sent to National Tuberculosis Reference Laboratory of Public Health Institution of Türkiye for susceptibility testing to first and second line drugs. Mutation and wild-type determination were studied by "Line Probe Assay (LPA)" method to investigate the susceptibility of the isolates to isoniazid, rifampicin, fluoroquinolone and aminoglycoside/cyclic peptide. The results obtained from the patients were collected and evaluated retrospectively from the records. Growth was observed in 18 samples out of 171 sputum samples from 67 patients; 13 isolates were detected as MDR-TB while one isolate was detected as mono RR-TB. The rate of mono RR-TB was 1.5% and the rate of MDR-TB was 19.4%. MUT3 causing rifampicin resistance was detected in 17.9% of the patients, katG/MUT1 causing isoniazid resistance in 17.9% and WT loss causing aminoglycoside/cyclic peptide resistance were detected in 19.4% of the patients. Neither fluoroquinolone resistance nor a mutation leading to fluoroquinolone resistance was detected in the study. When the sputum samples taken from the patients living in Northern Syria were examined, the frequency of MDR-TB was found to be quite high. MDR-TB, which is an important public health problem, was found at high rates due to the internal turmoil in the region and poor accessibility to health services. Since the gene mutations causing drug resistance with the LPA method differ with the conducted studies, it is important to evaluate the dominant gene mutations for determining the TB treatment strategies in the region.

Investigation of O25b-ST131 Clone Frequency and Presence of Carbapenem and Fosfomycin Resistance Genes in Escherichia coli Isolates: First Detection of fosA3 from Escherichia coli O25bSTf131 Clone from Türkiye

Escherichia coli ST131 clone was first reported in 2008 and defined as a 'high-risk pandemic clone'. This clone plays a critical role in the spread of antimicrobial resistance worldwide. It was reported in many studies that the E.coli ST131 clone is widespread worldwide and can carry virulence and antibiotic resistance genes. E.coli ST131 clone is associated with a fluoroquinolone and broad-spectrum cephalosporin resistance. The agents used in the treatment of infections caused by the E.coli ST131 clone are limited. For this reason, monitoring the resistance status of these limited agents is crucial. In this study, we aimed to investigate the prevalence of the O25b-ST131 clone, and the presence of carbapenem and fosfomycin resistance genes in fluoroquinolone-resistant E.coli isolates isolated from mid-stream urine samples. For the detection of the O25b-ST131 clone in fluoroquinolone-resistant E.coli isolates, amplification was performed with primers O25pabBspe-F and O25pabBspe-R. For the determination of resistance genes, carbapenem resistance genes blaNDM, blaVIM, blaKPC, blaIMP, and blaOXA-48 in carbapenem resistant isolates and plasmid-mediated fosfomycin resistance genes fosA3 and fosC2 in fosfomycin resistant isolates were investigated by multiplex PCR method. According to PCR results, the prevalence of E.coli O25b-ST131 isolates was 51.2%. Carbapenem resistance rate was 3.41%, and fosfomycin resistance rate was 3.41% in fluoroquinolone-resistant E.coli isolates. Carbapenem and fosfomycin resistance rates in E.coli O25bST131 isolates were determined as 0.83% and 2.5%, respectively. At least one of the carbapenem-resistance genes (blaNDM and/or blaOXA-48) was detected in six of the eight carbapenem-resistant isolates. Fosfomycin resistance gene fosA3 was seen in four of eight fosfomycin-resistant isolates. fosC2 gene was not detected in any of the isolates. In addition, the plasmid-mediated fosfomycin resistance gene fosA3 was detected in an E.coli O25b-ST131 isolate, and this result was confirmed by sequence analysis. To the best of our knowledge, this is the second report about fosA3 positivity in E.coli ST131 isolates from the world and the first reported from Europe and Türkiye. As a result, approximately half of the fluoroquinolone-resistant E.coli isolates were identified as E.coli ST131 clones. Despite the high rate of this clone, the carbapenem and fosfomycin resistance rates are still relatively low, which is pleasing for the future of treatment. However, it should not be forgotten that resistance rates and the prevalence of resistance genes should be constantly monitored.

Decoding a cryptic mechanism of metronidazole resistance among globally disseminated fluoroquinolone-resistant Clostridioides difficile

Severe outbreaks and deaths have been linked to the emergence and global spread of fluoroquinolone-resistant Clostridioides difficile over the past two decades. At the same time, metronidazole, a nitro-containing antibiotic, has shown decreasing clinical efficacy in treating C. difficile infection (CDI). Most metronidazole-resistant C. difficile exhibit an unusual resistance phenotype that can only be detected in susceptibility tests using molecularly intact heme. Here, we describe the mechanism underlying this trait. We find that most metronidazole-resistant C. difficile strains carry a T-to-G mutation (which we term PnimBG) in the promoter of gene nimB, resulting in constitutive transcription. Silencing or deleting nimB eliminates metronidazole resistance. NimB is related to Nim proteins that are known to confer resistance to nitroimidazoles. We show that NimB is a heme-dependent flavin enzyme that degrades nitroimidazoles to amines lacking antimicrobial activity. Furthermore, occurrence of the PnimBG mutation is associated with a Thr82Ile substitution in DNA gyrase that confers fluoroquinolone resistance in epidemic strains. Our findings suggest that the pandemic of fluoroquinolone-resistant C. difficile occurring over the past few decades has also been characterized by widespread resistance to metronidazole.

Diagnostic low-dose X-ray radiation induces fluoroquinolone resistance in pathogenic bacteria

Purpose The crisis of antibiotic resistance has been attributed to the overuse or misuse of these medications. However, exposure of bacteria to physical stresses such as X-ray radiation, can also lead to the development of resistance to antibiotics. The present study aimed to investigate the effect of exposure to diagnostic low-dose X-ray radiation on the bacterial response to antibiotics in two pathogenic bacteria, including the Gram-positive Staphylococcus aureus and Gram-negative Salmonella enteritidis. Methods The bacterial strains were exposed to diagnostic X-ray doses of 5 and 10 mGy, which are equivalent to the doses delivered to patients during conventional radiography X-ray examinations in accordance with the European guidelines on quality criteria for diagnostic radiographic images. Following exposure to X-ray radiation, the samples were used to estimate bacterial growth dynamics and perform antibiotic susceptibility tests. Results The results indicate that exposure to diagnostic low-dose X-ray radiation increased the number of viable bacterial colonies of both Staphylococcus aureus and Salmonella enteritidis and caused a significant change in bacterial susceptibility to antibiotics. For instance, in Staphylococcus aureus, the diameter of the inhibition zones for marbofloxacin decreased from 29.66 mm before irradiation to 7 mm after irradiation. A significant decrease in the inhibition zone was also observed for penicillin. In the case of Salmonella enteritidis, the diameter of the inhibition zone for marbofloxacin was 29 mm in unexposed bacteria but decreased to 15.66 mm after exposure to 10 mGy of X-ray radiation. Furthermore, a significant decrease in the inhibition zone was detected for amoxicillin and amoxicillin/clavulanic acid (AMC). Conclusion It is concluded that exposure to diagnostic X-ray radiation can significantly alter bacterial susceptibility to antibiotics. This irradiation decreased the effectiveness of fluoroquinolone and β-lactam antibiotics. Specifically, low-dose X-rays made Staphylococcus aureus resistant to marbofloxacin and increased its resistance to penicillin. Similarly, Salmonella Enteritidis became resistant to both marbofloxacin and enrofloxacin, and showed reduced sensitivity to amoxicillin and AMC.

High Burden of Intestinal Colonization With Antimicrobial-Resistant Bacteria in Chile: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study.

Antimicrobial resistance is a global threat, heavily impacting low- and middle-income countries. This study estimated antimicrobial-resistant gram-negative bacteria (GNB) fecal colonization prevalence in hospitalized and community-dwelling adults in Chile before the coronavirus disease 2019 pandemic. From December 2018 to May 2019, we enrolled hospitalized adults in 4 public hospitals and community dwellers from central Chile, who provided fecal specimens and epidemiological information. Samples were plated onto MacConkey agar with ciprofloxacin or ceftazidime added. All recovered morphotypes were identified and characterized according to the following phenotypes: fluoroquinolone-resistant (FQR), extended-spectrum cephalosporin-resistant (ESCR), carbapenem-resistant (CR), or multidrug-resistant (MDR; as per Centers for Disease Control and Prevention criteria) GNB. Categories were not mutually exclusive. A total of 775 hospitalized adults and 357 community dwellers were enrolled. Among hospitalized subjects, the prevalence of colonization with FQR, ESCR, CR, or MDR-GNB was 46.4% (95% confidence interval [CI], 42.9-50.0), 41.2% (95% CI, 37.7-44.6), 14.5% (95% CI, 12.0-16.9), and 26.3% (95% CI, 23.2-29.4). In the community, the prevalence of FQR, ESCR, CR, and MDR-GNB colonization was 39.5% (95% CI, 34.4-44.6), 28.9% (95% CI, 24.2-33.6), 5.6% (95% CI, 3.2-8.0), and 4.8% (95% CI, 2.6-7.0), respectively. A high burden of antimicrobial-resistant GNB colonization was observed in this sample of hospitalized and community-dwelling adults, suggesting that the community is a relevant source of antibiotic resistance. Efforts are needed to understand the relatedness between resistant strains circulating in the community and hospitals.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

Development of antisense peptide-peptide nucleic acids against fluoroquinolone-resistant Escherichia coli.

Fluoroquinolones (FQs) are potent and broad-spectrum antibiotics commonly used to treat MDR bacterial infections, but bacterial resistance to FQs has emerged and spread rapidly around the world. The mechanisms for FQ resistance have been revealed, including one or more mutations in FQ target genes such as DNA gyrase (gyrA) and topoisomerase IV (parC). Because therapeutic treatments for FQ-resistant bacterial infections are limited, it is necessary to develop novel antibiotic alternatives to minimize or inhibit FQ-resistant bacteria. To examine the bactericidal effect of antisense peptide-peptide nucleic acids (P-PNAs) that can block the expression of DNA gyrase or topoisomerase IV in FQ-resistant Escherichia coli (FRE). A set of antisense P-PNA conjugates with a bacterial penetration peptide were designed to inhibit the expression of gyrA and parC and were evaluated for their antibacterial activities. Antisense P-PNAs, ASP-gyrA1 and ASP-parC1, targeting the translational initiation sites of their respective target genes significantly inhibited the growth of the FRE isolates. In addition, ASP-gyrA3 and ASP-parC2, which bind to the FRE-specific coding sequence within the gyrA and parC structural genes, respectively, showed selective bactericidal effects against FRE isolates. Our results demonstrate the potential of targeted antisense P-PNAs as antibiotic alternatives against FQ-resistance bacteria.

The fitness cost of horizontally transferred and mutational antimicrobial resistance in Escherichia coli.

Antimicrobial resistance (AMR) in bacteria implies a tradeoff between the benefit of resistance under antimicrobial selection pressure and the incurred fitness cost in the absence of antimicrobials. The fitness cost of a resistance determinant is expected to depend on its genetic support, such as a chromosomal mutation or a plasmid acquisition, and on its impact on cell metabolism, such as an alteration in an essential metabolic pathway or the production of a new enzyme. To provide a global picture of the factors that influence AMR fitness cost, we conducted a systematic review and meta-analysis focused on a single species, Escherichia coli. By combining results from 46 high-quality studies in a multilevel meta-analysis framework, we find that the fitness cost of AMR is smaller when provided by horizontally transferable genes such as those encoding beta-lactamases, compared to mutations in core genes such as those involved in fluoroquinolone and rifampicin resistance. We observe that the accumulation of acquired AMR genes imposes a much smaller burden on the host cell than the accumulation of AMR mutations, and we provide quantitative estimates of the additional cost of a new gene or mutation. These findings highlight that gene acquisition is more efficient than the accumulation of mutations to evolve multidrug resistance, which can contribute to the observed dominance of horizontally transferred genes in the current AMR epidemic.

Phylogeny, Virulence, and Antimicrobial Resistance Gene Profiles of Enterococcus faecium Isolated from Australian Feedlot Cattle and Their Significance to Public and Environmental Health.

The extent of similarity between E. faecium strains found in healthy feedlot beef cattle and those causing extraintestinal infections in humans is not yet fully understood. This study used whole-genome sequencing to analyse the antimicrobial resistance profile of E. faecium isolated from beef cattle (n = 59) at a single feedlot and compared them to previously reported Australian isolates obtained from pig (n = 60) and meat chicken caecal samples (n = 8), as well as human sepsis cases (n = 302). The E. faecium isolated from beef cattle and other food animal sources neither carried vanA/vanB responsible for vancomycin nor possessed gyrA/parC and liaR/liaS gene mutations associated with high-level fluoroquinolone and daptomycin resistance, respectively. A small proportion (7.6%) of human isolates clustered with beef cattle and pig isolates, including a few isolates belonging to the same sequence types ST22 (one beef cattle, one pig, and two human isolates), ST32 (eight beef cattle and one human isolate), and ST327 (two beef cattle and one human isolate), suggesting common origins. This provides further evidence that these clonal lineages may have broader host range but are unrelated to the typical hospital-adapted human strains belonging to clonal complex 17, significant proportions of which contain vanA/vanB and liaR/liaS. Additionally, none of the human isolates belonging to these STs contained resistance genes to WHO critically important antimicrobials. The results confirm that most E. faecium isolated from beef cattle in this study do not pose a significant risk for resistance to critically important antimicrobials and are not associated with current human septic infections.

Different stages of the infection cycle are enriched for Campylobacter strains with distinct phenotypes and levels of fluoroquinolone resistance.

Campylobacter species are the leading cause of bacterial diarrhoea worldwide and consumption of contaminated chicken meat is the most common route of infection. Chickens can be infected with multiple strains of Campylobacter and during the infection cycle this pathogen must survive a wide variety of environments. Numerous studies have reported a high degree of genetic variability in this pathogen that can use antigenic and phase variation to alter the expression of key phenotypes. In this study the phenotypic profile of isolates from freshly slaughtered chickens, chicken products in the supermarket and stool samples from infected patients were compared to identify phenotypic changes during the passage of the bacteria through the infection cycle. Isolates from different stages of the infection cycle had altered phenotypic profiles with isolates from human stool samples showing a lower ability to form a biofilm and an increased ability to associate with epithelial cells in vitro. Resistance to fluoroquinolones was found in all cohorts but at a much higher occurrence (94%) in isolates from supermarket chicken. Isolates displaying high levels of resistance to fluoroquinolones also were more likely to display a higher tolerance to growth in the presence of oxygen. In conclusion, isolates with specific phenotypes appear to be overly represented at different stages of the infection cycle suggesting that environmental stresses may be enriched for strains with these phenotypes.

Molecular detection of Helicobacter pylori and its genotypic antimicrobial resistance patterns in dyspeptic Mozambican patients

AbstractBackgroundHelicobacter pylori strains show a high level of genotypic diversity and express several genes that contribute to their pathogenicity and resistance. In Mozambique, there is lack of information regarding its resistance pattern to antibiotics. In this study, we aimed to investigate the prevalence of H. pylori and its genotypic resistance to clarithromycin, metronidazole, and fluoroquinolones in Mozambican dyspeptic patients. Since appropriate eradication should be based on the local resistance rate, our data will guide clinicians in choosing the best drugs for the effective treatment of H. pylori‐infected patients.MethodsThis is a cross‐sectional descriptive study conducted between June 2017 and June 2020, in which 171 dyspeptic patients were recruited, and through upper gastrointestinal endoscopy, gastric biopsies were collected from those patients. Polymerase chain reaction was performed for the detection of H. pylori and its resistance mechanisms to clarithromycin (23S rRNA), metronidazole (rdxA), and fluoroquinolones (gyrA); mutations conferring resistance to these antibiotics were investigated by sequencing 23S rRNA, rdxA, and gyrA genes.ResultsOf the 171 samples tested, H. pylori was detected in 56.1% (96/171). The clarithromycin resistance rate was 10.4% (the responsible mutations were A2142G and A2143G), the metronidazole resistance rate was 55.2% (4 types of mutations responsible for metronidazole resistance were identified which include, D59N, R90K, H97T, and A118T. However, in many cases, they appeared in combination, with D59N + R90K + A118T being the most frequent combination), and the fluoroquinolones resistance rate was 20% (the responsible mutations were N87I and D91G).ConclusionH. pylori infection remains common in dyspeptic Mozambican patients. High resistance to metronidazole and fluoroquinolones requires continuous monitoring of antibiotic resistance and adaptation of therapy to eradicate this infection.

Molecular epidemiology of antibiotic-resistant Escherichia coli among clinical samples isolated in Azerbaijan, Iran.

The immediate emergence of resistant bacteria poses an increasingly growing problem to human society and the increasing prevalence of antibiotic resistance in Escherichia coli strains is one of the most important health problems. This study aimed to review the molecular epidemiology of drug resistance among clinical isolates of E. coli in north-west portion of Iran Azerbaijan. A complete of 219 clinical isolates of E. coli had been collected from the various clinical samples. The disk diffusion and agar dilution assays were used to determine antimicrobial susceptibility. The presence of antibiotics resistance genes was carried out by the PCR method. The highest susceptibility was shown to imipenem (3%) and fosfomycin (3%), and the most antibiotic resistance was presented to ampicillin (99%). The highest frequent ESBL gene among isolates was bla CTXM-15 in 70% followed by blaCMY-2 in 67%, and blaTEM-1 in 46%. The most common fluoroquinolone (FQ) resistance genes were oqxB (34%), followed by oqxA (25%), and qnrB (18%). The frequency of tetracycline resistance genes (tetA, tetB, tetC, and tetD) were detected in 24.8%, 31.6%, 1.8%, and 4.2%, respectively. The highest frequent genes to fosfomycin were fosA 10%, fosA3 30%, fosC 40%, and fosX 20%. The dominant founded aminoglycosides resistant genes were armA (12.96%) and npmA (4.93%). The prevalence of antibiotics resistance in the tested E. coli isolates was high in Azerbaijan, Iran and these findings showed that E. coli is one of the major drug-resistant pathogens.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Retreatment after loss to follow-up reduces mortality in patients with multidrug/rifampicin-resistant tuberculosis.

This study evaluated the risk factors of long-term mortality in patients with multidrug/rifampicin-resistant tuberculosis (MDR/RR-TB) in South Korea who were lost to follow-up (LTFU). This was a retrospective longitudinal follow-up study using an integrated database constructed by data linkage of the three national databases, which included 7226 cases of MDR/RR-TB notified between 2011 and 2017 in South Korea. Post-treatment outcomes of patients who were LTFU were compared with those of patients who achieved treatment success. Of the 7226 MDR/RR-TB cases, 730 (10.1%) were LTFU. During a median follow-up period of 4.2 years, 101 (13.8%) of the LTFU patients died: 25 deaths (3.4%) were TB related and 76 (10.4%) were non-TB related. In the LTFU group, the adjusted hazard ratio (aHR) of all-cause mortality (aHR 2.50, 95% CI 1.99-3.15, p<0.001), TB-related mortality (aHR 5.38, 95% CI 3.19-9.09, p<0.001) and non-TB-related mortality (HR 2.21, 95% CI 1.70-2.87, p<0.001) was significantly higher than that in the treatment success group. Independent risk factors for all-cause mortality in the LTFU group were age >55 years, fluoroquinolone resistance, cancer and no retreatment. In the LTFU patients who did not receive retreatment, the risk of non-TB-related mortality (aHR 5.00, 95% CI 1.53-16.37, p=0.008) and consequent all-cause mortality (aHR 2.18, 95% CI 1.08-4.40, p=0.030) was significantly higher than that of patients who received retreatment. Non-TB-related mortality was the main cause of death and might be reduced by retreatment in LTFU patients with MDR/RR-TB.

Characterization of Genetic Mutations in Multi-Drug-Resistant Isolates of Mycobacterium tuberculosis Bacilli Conferring Resistance to a Second-Line Anti-tuberculosis Drug.

Multi-drug-resistant tuberculosis (MDR-TB) has become a major public health concern globally. Mutations in first- and second-line drug targets such as katG, inhA, rpoB, rrs, eis, gyrA, and gyrB have been associated with drug resistance. Monitoring predominant mutations in the MDR-TB patient population is essential to monitor and devise future therapeutic regimes. The present study is aimed to characterize genetic mutations in MDR isolates of Mycobacterium tuberculosis (MTB)bacilli conferring resistance to a second-line anti-tuberculosis drug in the Eastern Indian population. This cross-sectional study was conducted in the Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, and in the Tuberculosis Demonstration & Training Centre, Agamkuan, Patna. A total of 3270 patients suspected to have MDR-TB were recruited in the study. Two sputum samples, one on the spot, and the other in the morning were collected from each patient and the diagnosis ofrifampicin-sensitive (RS)/rifampicin-resistant (RR/MDR) TB was done by Gene-Xpert test. One hundred fifty RS-TB samples and 150 RR/MDR-TBsamples were considered for line probe assay (LPA). RSsamples were subjected to first-line LPA using Genotype® MTBDR Plus ver 2.0 and RR/MDRsamples were considered for second-line LPA using Genotype®MTBDRslver 2.0. All sputum samples were subjected to sputum smear microscopy using the Ziehl-Neelsen staining method.Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 26.0 (IBM Corp. Armonk, NY) and R (version 4.1; R Core Team 2021). In the present study, out of 3270 patients, we detected RR/MDR-TB in 235 patients (7.19%),RS-TB in 812 patients (24.83%), the rest of the patients negative for MTB (2223, 67.98%). Out of 150 RR/MDR-TB sputum samples tested, resistance to fluoroquinolone (FQ) was observed in 41 samples. The selected patients had predominantly FQ resistance due to the gyrA gene mutations (97.56%, n=40) compared to the gyrB gene mutations (2.44%, n=1). We observed >60% of the mutations in the gyrA gene in codon 94 (MUT3C (D94G), MUT3A (D94A), and MUT3D (D94H).In addition, we found the mutations MUT1 (A90V) and MUT2 (S91P)in the codons 90 and 91 of the gyrA gene in the considered MTB patient population. The identified genes can be further validated to be considered as therapeutic targets, but more therapeutics and advanced strategies should be applied in the management of MTB.

Clinically relevant antibiotic resistance in Escherichia coli from black kites in southwestern Siberia: a genetic and phenotypic investigation.

Wild birds including raptors can act as vectors of clinically relevant bacteria with antibiotic resistance. The aim of this study was to investigate the occurrence of antibiotic-resistant Escherichia coli in black kites (Milvus migrans) inhabiting localities in proximity to human-influenced environments in southwestern Siberia and investigate their virulence and plasmid contents. A total of 51 E. coli isolates mostly with multidrug resistance (MDR) profiles were obtained from cloacal swabs of 35 (64%, n = 55) kites. Genomic analyses of 36 whole genome sequenced E. coli isolates showed: (i) high prevalence and diversity of their antibiotic resistance genes (ARGs) and common association with ESBL/AmpC production (27/36, 75%), (ii) carriage of mcr-1 for colistin resistance on IncI2 plasmids in kites residing in proximity of two large cities, (iii) frequent association with class one integrase (IntI1, 22/36, 61%), and (iv) presence of sequence types (STs) linked to avian-pathogenic (APEC) and extra-intestinal pathogenic E. coli (ExPEC). Notably, numerous isolates had significant virulence content. One E. coli with APEC-associated ST354 carried qnrE1 encoding fluoroquinolone resistance on IncHI2-ST3 plasmid, the first detection of such a gene in E. coli from wildlife. Our results implicate black kites in southwestern Siberia as reservoirs for antibiotic-resistant E. coli. It also highlights the existing link between proximity of wildlife to human activities and their carriage of MDR bacteria including pathogenic STs with significant and clinically relevant antibiotic resistance determinants. IMPORTANCE Migratory birds have the potential to acquire and disperse clinically relevant antibiotic-resistant bacteria (ARB) and their associated antibiotic resistance genes (ARGs) through vast geographical regions. The opportunistic feeding behavior associated with some raptors including black kites and the growing anthropogenic influence on their natural habitats increase the transmission risk of multidrug resistance (MDR) and pathogenic bacteria from human and agricultural sources into the environment and wildlife. Thus, monitoring studies investigating antibiotic resistance in raptors may provide essential data that facilitate understanding the fate and evolution of ARB and ARGs in the environment and possible health risks for humans and animals associated with the acquisition of these resistance determinants by wildlife.

Targeted Antimicrobial Prophylaxis with Cefmetazole Based on Presence of Fluoroquinolone-Resistant Isolates to Prevent Post-Prostate Biopsy Infectious Complications

Fluoroquinolones (FQs) have been traditionally used for prophylaxis against bacterial infection. However, the rapid emergence of FQ-resistant Escherichia coli due to overuse and misuse have resulted in an increase in post-biopsy infections. We requested 723 patients undergoing transrectal or transrectal plus transperineal targeted prostate biopsy to provide preprocedure rectal swabs. The rectal swabs were plated onto deoxycholate hydrogen sulfate lactose agar culture and FQ resistance tests were conducted using the disc diffusion method following the guidelines of the Clinical and Laboratory Standards Institute. All patients undergoing biopsy were given a 1.0 g intravenous injection of cefmetazole (CMZ) 30 min before and 12 h after biopsy. Patients with FQ-resistant organisms received an additional 1.0 g intravenous injection of CMZ every 12 h for an additional 1.5 days, while those without FQ-resistant organisms received levofloxacin 500 mg for 4 days. We evaluated infectious symptoms during the 30 days after the biopsy. We also evaluated the incidence of acute prostatitis within 7 days after the biopsy and isolation rates of FQ-resistant strains. A total of 289 patients (40%) had FQ-resistant isolates on rectal swabs. The overall infectious complication rate was 0.69%. Two patients with FQ-resistant isolates and three patients without them experienced infectious episodes. One patient with FQ-resistant isolates and two patients without them suffered acute prostatitis. The difference in the rates of infectious complication and acute prostatitis rates between FQ-resistant and FQ-susceptible carriers were not significant (p = 1.0 and 1.0, respectively). Post-biopsy sepsis was identified in one patient (0.14%) who had FQ-resistant Escherichia coli. Targeted antimicrobial prophylaxis with cefmetazole based on presence of FQ-resistant isolates on rectal swabs may prevent post-prostate biopsy infectious complications, especially in geographic lesions with a high incidence of FQ-resistant strains in rectal flora.

Cross-media migration behavior of antibiotic resistance genes (ARGs) from municipal wastewater treatment systems (MWTSs): Fugitive characteristics, sharing mechanisms, and aerosolization behavior

The municipal wastewater treatment systems (MWTSs) are the leading enrichment site of antibiotic resistance genes (ARGs), the occurrence of which in sewage and sludge significantly influences the ARGs burden of aerosols. However, the migration behavior and impact factors of ARGs in gas–liquid–solid phase are still unclear. This study collected gas (aerosol), liquid (sewage), and solid (sludge) samples from three MWTSs to explore the cross-media transport behavior of ARGs. The results showed that the main ARGs detected in the solid–gas-liquid phase were consistent, constituting the central antibiotic resistance system of MWTSs. Multidrug resistance genes dominated cross-media transmission (average relative abundance is 42.01 %). Aminocoumarin, fluoroquinolone, and aminoglycoside resistance genes (aerosolization index of 1.260, 1.329, and 1.609, respectively) were prone to migrating from the liquid to gas phase, resulting in long-distance transmission. Environmental factors (mainly temperature and wind speed), water quality index (mainly COD), and heavy metals may be the key factors affecting the trans-media migration of ARGs between the liquid, gas, and solid phase. Based on partial least squares path modeling (PLS-PM), the migration of ARGs in gas phase is primarily influenced by ARGs' aerosolization potential in liquid and solid phase, while heavy metals indirectly influences almost all categories of ARGs. Impact factors aggravated the migration of ARGs in MWTSs through co-selection pressure. This study clarified the key pathways and impact factors that form the cross-media migration behavior of ARGs, which can more specifically control ARGs pollution from different media.