Levofloxacin-resistant Isolates Research Articles

Antibacterial and antibiofilm activities of diclofenac against levofloxacin-resistant Stenotrophomonas maltophilia isolates; emphasizing repurposing of diclofenac

Background and Objectives: Stenotrophomonas maltophilia is an opportunistic pathogen causing nosocomial infections. Diclofenac is an anti-inflammatory drug that is considered a non-antibiotic drug. This study assessed the antibacterial and antibiofilm effects of diclofenac and levofloxacin/diclofenac combination against levofloxacin resistant isolates.
 Materials and Methods: Minimum inhibitory concentration was determined using broth microdilution method for levo- floxacin, diclofenac, and levofloxacin/diclofenac combination. Biofilm forming capacity and biofilm inhibition assay were determined. Relative gene expression was measured for efflux pump genes; smeB, and smeF genes and biofilm related genes rmlA, spgM, and rpfF without and with diclofenac and the combination.
 Results: Diclofenac demonstrated MIC of 1 mg/ml. The combination-with ½ MIC diclofenac- showed synergism where levofloxacin MIC undergone 16-32 fold decrease. All the isolates that overexpressed smeB and smeF showed a significant decrease in gene expression in presence of diclofenac or the combination. The mean percentage inhibition of biofilm for- mation with diclofenac and the combination was 40.59% and 46.49%, respectively. This agreed with biofilm related genes expression investigations.
 Conclusion: Diclofenac showed an antibacterial effect against Stenotrophomonas maltophilia. The combination showed in-vitro synergism, significant reduction in biofilm formation and in the relative level of gene expression. Furthermore, it can potentiate the levofloxacin activity or revert its resistance.

Molecular characterization and antimicrobial resistance of Streptococcus agalactiae isolated from pregnant women in Japan, 2017–2021

ObjectivesThis study aimed to elucidate the molecular characteristics and antimicrobial resistance of Streptococcus agalactiae (group B streptococcus, GBS) colonizing pregnant women in Japan. MethodsGBS isolates obtained from screening of pregnant women from 2017 to 2021 were analyzed for capsular serotype, sequence type (ST), and antimicrobial susceptibility. For levofloxacin-resistant isolates, mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA, gyrB, and parC genes were analyzed. ResultsSeventy-six GBS isolates were recovered from 1090 women (isolation rate: 7.0%). Of the 76 isolates, serotype III (31.6%) was the most prevalent, followed by V (19.7%), Ia (17.1%), and Ib (10.5%). Among the 22 STs identified, capsular serotype III/ST335-clonal complex (CC) 19 lineage was dominant (13.2%), followed by Ia/ST23, III/ST17, and V/ST1. Levofloxacin resistance was detected in 15.8% (n=12) of all the isolates, with serotype Ib being the most common. Most levofloxacin resistant isolates belonged to serotype Ib/CC10 or serotype V/CC19, with double mutations in the QRDRs, Ser81Leu in GyrA and Ser79Phe in ParC. ConclusionsThe present study indicates the prevalence of the serotype III/ST335 (CC19) lineage, and the spread of serotype Ib/CC10 and serotype V/CC19 lineages, which are responsible for levofloxacin resistance in colonizing GBS in pregnant women in Japan.

ROLE OF RND-EFFLUX PUMP IN LEVOFLOXACIN RESISTANCE AMONG ACINETOBACTER BAUMANNII

In this study out of 130 different clinical specimens, 70 Acinetobacter baumannii were collected from hospitalized patients. Then; all the isolates were characterized using biochemical tests and confirmed by using the Vitek II compact system. All isolates of Acinetobacter baumannii were subjected to the disc diffusion method to determine it's a susceptibility to 8 types of antibiotic disc, and the result showed these bacteria were resistant to Cefepime, Imipenem, trimethoprim, Piperacillin, tetracycline, Amikacin, ampicillin-sulbactam and levofloxacin. According to bacterial susceptibility test, all isolated were showed highly resistance to Trimethoprim (88.57%) and the lower resistance to tetracycline (47.14%). Minimum inhibitory concentration was determined only for levofloxacin resistant isolates of Acinetobacter baumannii using the broth dilution method. MIC shows these bacteria were resistant to levofloxacin in concentration ranging between 32µg/100µl to 128µg/100µl. The relation between adeIK and levofloxacin resistance was calculated by determining gene expression using real-time PCR. The result showed the mean value of adeI and adeK before exposing bacteria to levofloxacin was 2.3 and 0.42 respectively while the mean value of adeI and adeK after exposing bacteria to levofloxacin was 3.15 and 3.29 respectively. This study indicates that adeI has a non-significant role (p-value=0.11) in providing resistance to levofloxacin, while adeK plays a significant role (p-value= 0.008) in providing resistance to levofloxacin.

In vitro activity of delafloxacin against highly levofloxacin-resistant invasive isolates of Streptococcus pneumoniae

IntroductionWe report the activity of delafloxacin, a new fluoroquinolone with high affinity for both topoisomerase IV and DNA gyrase, against highly-levofloxacin-resistant invasive strains of Streptococcus pneumoniae. MethodsA total of 173 highly-levofloxacin-resistant (MIC>32mg/L) S. pneumoniae invasive isolates were studied. The strains were isolated from blood (n=162) and other sterile fluids (n=11). Serotyping was performed by the Pneumotest-Latex and Quellung reaction.Delafloxacin, levofloxacin, penicillin, cefotaxime, erythromycin and vancomycin MICs were determined by the gradient diffusion method following EUCAST guidelines and breakpoints. ResultsAmong the isolates, 32.9% were penicillin non-susceptible, 19.7% cefotaxime non-susceptible, and 76.9% erythromycin resistant. All were susceptible to vancomycin. Delafloxacin MIC50 and MIC90 (mg/L) values were 0.064 and 0.12, respectively; 60% (15/25) of serotype 9V isolates showed delafloxacin MICs≥0.12mg/L. ConclusionsDelafloxacin was very active against highly-levofloxacin-resistant invasive isolates of S. pneumoniae. Isolates belonging to serotype 9V showed higher delafloxacin MIC values.

PCV-15 and PPSV-23 coverage of invasive and respiratory tract Streptococcus pneumoniae, including MDR and XDR isolates: CANWARD 2007-20.

To compare the proportion of invasive and respiratory tract isolates of Streptococcus pneumoniae, including MDR and XDR strains, that demonstrated PCV-15 and PPSV-23 serotypes in Canada from 2007 to 2020. The CANWARD study collected 2984 S. pneumoniae isolates from 2007 to 2020 (1054 invasive, 1930 respiratory). Serotyping was performed using the Quellung reaction. Antimicrobial susceptibility testing was performed using CLSI methods. MDR/XDR was defined as resistance to ≥3/≥5 antimicrobial classes, respectively. Overall, the proportion of vaccine serotypes demonstrating a PCV-15/PPSV-23 serotype was significantly higher in blood isolates (54.6%/76.2%, respectively) than respiratory isolates (38.9%/55.3%; P < 0.0001). Similarly, PCV-15 and PPSV-23 vaccine coverage was higher for blood isolates for all demographic categories, including both genders, all regions and all age groups (P ≤ 0.0213). PCV-15/PPSV-23 coverage was also significantly higher for blood isolates demonstrating clarithromycin resistance (60.4/75.1% blood, 47.8/57.4% respiratory; P ≤ 0.009) and penicillin resistance (68.9/63.0% blood, 45.2/43.0% respiratory; P < 0.0001) and trimethoprim/sulfamethoxazole-resistant isolates for PPSV-23 only (82.6% blood, 64.3% respiratory; P = 0.0057). Vaccine coverage was numerically higher but not significantly different between specimen source for children <2 years of age, as well as ceftriaxone-, doxycycline- and levofloxacin-resistant isolates. PCV-15/PPSV-23 vaccine coverage for MDR isolates (61.8%/67.3% blood, 52.2%/56.2% respiratory) and XDR isolates (93.3% blood, 89.6% respiratory for both vaccines) was not significantly different between specimen sources. PCV-15 and PPSV-23 serotype coverage is generally greater for blood versus respiratory isolates but not for MDR and XDR isolates. Continued pneumococcal surveillance is warranted to determine future trends in vaccine coverage, serotype distribution and antimicrobial susceptibilities under the pressure of vaccine use.

In vitro activity of delafloxacin against highly levofloxacin-resistant invasive isolates of Streptococcus pneumoniae

IntroductionWe report the activity of delafloxacin, a new fluoroquinolone with high affinity for both topoisomerase IV and DNA gyrase, against highly-levofloxacin-resistant invasive strains of Streptococcus pneumoniae. MethodsA total of 173 highly-levofloxacin-resistant (MIC>32mg/L) S. pneumoniae invasive isolates were studied. The strains were isolated from blood (n=162) and other sterile fluids (n=11). Serotyping was performed by the Pneumotest-Latex and Quellung reaction.Delafloxacin, levofloxacin, penicillin, cefotaxime, erythromycin and vancomycin MICs were determined by the gradient diffusion method following EUCAST guidelines and breakpoints. ResultsAmong the isolates, 32.9% were penicillin non-susceptible, 19.7% cefotaxime non-susceptible, and 76.9% erythromycin resistant. All were susceptible to vancomycin. Delafloxacin MIC50 and MIC90 (mg/L) values were 0.064 and 0.12, respectively; 60% (15/25) of serotype 9V isolates showed delafloxacin MICs≥0.12mg/L. ConclusionsDelafloxacin was very active against highly-levofloxacin-resistant invasive isolates of S. pneumoniae. Isolates belonging to serotype 9V showed higher delafloxacin MIC values.

Milk from healthy or infected cattle as a source of multi-drug resistant, AmpC β-lactamase-producing Escherichia coli

Drinking raw unpasteurized milk or products made from raw milk is a recent trend observed among the people due to their belief in enhancing nutritional properties and better taste. It poses a great risk of microbial transmission along with antibiotic resistance gene pool (extended spectrum beta-lactamase or ampC) among the consumers. The study was conducted to detect the occurrence of AmpC producing E. coli in raw milk samples collected from healthy and mastitic cattle in West Bengal. In total, 450 cow milk samples were collected from all the six agro-climatic zones of West Bengal. The samples were collected from healthy as well as infected cattle suffering with clinical or sub-clinical mastitis, irrespective of age and breed. The collected milk samples were tested with MAST INDEX reagent for the detection of sub-clinical mastitis. E. coli isolates obtained from cattle milk samples (n=450) were subjected to cefoxitin-cloxacillin double disc synergy test and PCR based confirmation of blaAmpC, class I integron and CITM genes. All the ACBL producing E. coli isolates were subjected for their resistance to other antimicrobials. All the levofloxacin and Co-Trimoxazole resistant ACBL-producing isolates were studied for PMQR, sul1, sul2, sul3 genes. E. coli was isolated from 205 collected milk samples (205/450, 45.6%). The phenotypical test detected 231/383 isolates (60.3%) were AMPC producers which were also found to possess blaAmpC. Comparison of ACBL-producing E. coli occurrence in cattle with mastitis, sub-clinical mastitis and healthy cattle revealed significantly higher occurrence rate in infected cattle than the healthy ones (p<0.05). Phenotypical antibiotic resistance pattern of the isolates showed resistance against penicillin-G (100%), cefoxitin (100%), ampicillin (42%), co-trimoxazole (13.5%) and levofloxacin (5.2%). Out of 30 co-trimoxazole resistant isolates identified, 25 (83%) and seven (30.4%) isolates possessed sul1 and sul2 gene, respectively, whereas, four (13.3%) isolates possessed both sul1 and sul2 genes. Three levofloxacin resistant isolates possessed qnrS gene. This is the first systemic study with substantial numbers of milk samples from healthy and infected cattle depicting occurrence of ACBL-producing E. coli and linkage present in the isolates between the genes encoding ACBL and quinolone or sulphonamide resistance.

Design and synthesis of novel desfluoroquinolone-aminopyrimidine hybrids as potent anti-MRSA agents with low hERG activity

Despite the fact that the introduction of a fluorine atom at the C-6 position has resulted in the evolution of fluoroquinolones, fluoroquinolone-induced cardiac toxicity has drawn considerable attention. In this context, desfluoroquinolone-based hybrids with involvement of C-7 aminopyrimidine functional group were designed and synthesized. The biological results showed majority of these hybrids still demonstrated potent anti-MRSA activity with MIC values between 0.38 and 1.5 μg/mL, despite the lack of the typical C-6 fluorine atom. Particularly, the most active B14 exhibited activities at submicromolar concentrations against a panel of MRSA strains including vancomycin-intermediate strains, levofloxacin-resistant isolates, and linezolid-resistant isolates, etc. As expected, it also displayed highly selective toxicity toward bacterial cells and low hERG inhibition. Further resistance development study indicated MRSA is unlikely to acquire resistance against B14. The docking study revealed that two hydrogen bonds were formed between the C-7 substituent and the surrounding DNA bases, which might contribute to overcome resistance by reducing the dependence on the magnesium-water bridge interactions with topoisomerase IV. These results indicate a promising strategy for developing new antibiotic quinolones to combat multidrug resistance and cardiotoxicity.

Frequency and patterns of second-line resistance conferring mutations among MDR-TB isolates resistant to a second-line drug from eSwatini, Somalia and Uganda (2014\u20132016)

BackgroundPulmonary tuberculosis is a leading cause of morbidity and mortality in developing countries. Drug resistance, a huge problem in this contagious disease, is driven by point mutations in the Mycobacterium tuberculosis genome however, their frequencies vary geographically and this affects applicability of molecular diagnostics for rapid detection of resistance. Here, we report the frequency and patterns of mutations associated with resistance to second-line anti-TB drugs in multidrug-resistant (MDR) M. tuberculosis isolates from eSwatini, Somalia and Uganda that were resistant to a second-line anti-TB drug.MethodsThe quinolone resistance determining region (QRDR) of gyrA/gyrB genes and the drug resistance associated fragment of rrs gene from 80 isolates were sequenced and investigated for presence of drug resistance mutations. Of the 80 isolates, 40 were MDR, of which 28 (70%) were resistant to a second-line anti-TB injectable drug, 18 (45%) were levofloxacin resistant while 12 (30%) were extensively drug resistant (XDR). The remaining 40 isolates were susceptible to anti-TB drugs. MIRU-VNTR analysis was performed for M/XDR isolates.ResultsWe successfully sub-cultured 38 of the 40 M/XDR isolates. The gyrA resistance mutations (Gly88Ala/Cys/Ala, Ala90Val, Ser91Pro, Asp94Gly/Asn) and gyrB resistance mutations (Asp500His, Asn538Asp) were detected in 72.2% (13/18) and 22.2% (4/18) of the MDR and levofloxacin resistant isolates, respectively. Overall, drug resistance mutations in gyrA/gyrB QRDRs occurred in 77.8% (14/18) of the MDR and levofloxacin resistant isolates. Furthermore, drug resistance mutations a1401g and g1484 t in rrs occurred in 64.3% (18/28) of the MDR isolates resistant to a second-line anti-TB injectable drug. Drug resistance mutations were not detected in drug susceptible isolates.ConclusionsThe frequency of resistance mutations to second-line anti-TB drugs in MDR-TB isolates resistant to second line anti-TB drugs from eSwatini, Somalia and Uganda is high, implying that rapid molecular tests are useful in detecting second-line anti-TB drug resistance in those countries. Relatedly, the frequency of fluoroquinolone resistance mutations in gyrB/QRDR is high relative to global estimates, and they occurred independently of gyrA/QRDR mutations implying that their absence in panels of molecular tests for detecting fluoroquinolone resistance may yield false negative results in our setting.

Substantial Contribution of SmeDEF, SmeVWX, SmQnr, and Heat Shock Response to Fluoroquinolone Resistance in Clinical Isolates of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an emerging multi-drug resistant opportunistic pathogen. Although fluoroquinolones (FQ) are still clinically valuable for the treatment of S. maltophilia infection, an increasing prevalence in FQ resistance has been reported. Overexpression of SmeDEF, SmeVWX, and SmQnr, and de-repressed expression of heat shock response are reported mechanisms responsible for FQ resistance in S. maltophilia; nevertheless, some of these mechanisms are identified from laboratory-constructed mutants, and it remains unclear whether they occur in clinical setting. In this study, we aimed to assess whether these mechanisms contribute substantially to FQ resistance in clinical isolates. Eighteen ciprofloxacin- and levofloxacin-resistant isolates were selected from 125 clinical isolates of S. maltophilia. The expression of smeE, smeW, and Smqnr genes of these isolates was investigated by RT-qPCR. The de-repressed heat shock response was assessed by rpoE expression at 37°C and bacterial viability at 40°C. The contribution of SmeDEF, SmeVWX, and SmQnr, and heat shock response to FQ resistance was evaluated by mutants construction and susceptibility testing. The results demonstrated that simply assessing the overexpression of SmeDEF, SmeVWX, and SmQnr by RT-qPCR may overestimate their contribution to FQ resistance. Simultaneous overexpression of SmeDEF and SmeVWX did not increase the resistance level to their common substrates, but extended the resistance spectrum. Moreover, the de-repressed expression of heat shock response was not observed to contribute to FQ resistance in the clinical isolates of S. maltophilia.

Levofloxacin Pharmacokinetics/Pharmacodynamics, Dosing, Susceptibility Breakpoints, and Artificial Intelligence in the Treatment of Multidrug-resistant Tuberculosis.

Levofloxacin is used for the treatment of multidrug-resistant tuberculosis; however the optimal dose is unknown. We used the hollow fiber system model of tuberculosis (HFS-TB) to identify 0-24 hour area under the concentration-time curve (AUC0-24) to minimum inhibitory concentration (MIC) ratios associated with maximal microbial kill and suppression of acquired drug resistance (ADR) of Mycobacterium tuberculosis (Mtb). Levofloxacin-resistant isolates underwent whole-genome sequencing. Ten thousands patient Monte Carlo experiments (MCEs) were used to identify doses best able to achieve the HFS-TB-derived target exposures in cavitary tuberculosis and tuberculous meningitis. Next, we used an ensemble of artificial intelligence (AI) algorithms to identify the most important predictors of sputum conversion, ADR, and death in Tanzanian patients with pulmonary multidrug-resistant tuberculosis treated with a levofloxacin-containing regimen. We also performed probit regression to identify optimal levofloxacin doses in Vietnamese tuberculous meningitis patients. In the HFS-TB, the AUC0-24/MIC associated with maximal Mtb kill was 146, while that associated with suppression of resistance was 360. The most common gyrA mutations in resistant Mtb were Asp94Gly, Asp94Asn, and Asp94Tyr. The minimum dose to achieve target exposures in MCEs was 1500 mg/day. AI algorithms identified an AUC0-24/MIC of 160 as predictive of microbiologic cure, followed by levofloxacin 2-hour peak concentration and body weight. Probit regression identified an optimal dose of 25 mg/kg as associated with >90% favorable response in adults with pulmonary tuberculosis. The levofloxacin dose of 25 mg/kg or 1500 mg/day was adequate for replacement of high-dose moxifloxacin in treatment of multidrug-resistant tuberculosis.

2001. Susceptibility of Aerococcus urinae to Fluoroquinolones: Broth Microdilution and Gradient Diffusion

Background Aerococcus urinae is an emerging urinary pathogen frequently identified by MALDI-TOF. It is generally susceptible to β-lactams, however, its susceptibility pattern to fluoroquinolones (FQ) remains variable. The goals of this study were (i) to evaluate the performance of the gradient diffusion method (Etest®) to determine FQ resistance compared with broth microdilution (BMD) and (ii) to estimate the resistance rate of A. urinae toward FQ in Quebec hospitals.MethodsTwo hundred seven consecutive isolates of A. urinae from urinary tract specimens originating from five hospitals in Quebec and Montreal were identified by MALDI-TOF (Vitek-MS and Bruker). All isolates were tested with the BMD and gradient diffusion methods. BMD was carried out in triplicate and was conducted in accordance with CLSI guidelines (M45-A3). Isolates with insufficient growth at 24 hours were reincubated and evaluated at 48 hours. The gradient diffusion method was carried out using Etest® strips on MH agar with 5% sheep blood.ResultsOf the 207 isolates of A. urinae, 52 (25%) gave uninterpretable results using the BMD method (insufficient growth = 20; trailing = 32). We obtained the following results for the remaining 155 isolates:Susceptible, n (%)Intermediate, n (%)Resistant, n (%)Ciprofloxacin105 (67%)16 (10%)35 (23%)Levofloxacin114 (74%)6 (4%)35 (23%)BMD readings were often complicated by noticeably poor growth. The categorical agreement of the Etest® was 83% for ciprofloxacin and 95% for levofloxacin. Four very major errors were identified in a preliminary manner on 11% (4/35) of the ciprofloxacin-resistant isolates and 11%(4/35) of the levofloxacin-resistant isolates. Agar dilution will be done to confirm these results.ConclusionIn our experience, the method recommended by the CLSI for A. urinae susceptibility testing of FQ presented several problems, including insufficient growth and difficult reading. The Etest® appears to be a promising method for susceptibility testing of FQ for urinary tract isolates, but will first require a further comparison with agar dilution methods. In our study, the rate of FQ non-susceptibility of A. urinae was 27% for levofloxacin and 33% for ciprofloxacin. Therefore, FQ cannot be empirically recommended for the treatment of urinary tract infections caused by A. urinae.Disclosures J. M. Leduc, Biomérieux: Investigator, Research grant.

Tetracycline and fluoroquinolone resistance in clinical Ureaplasma spp. and Mycoplasma hominis isolates in France between 2010 and 2015

As information on Ureaplasma spp. and Mycoplasma hominis resistance is currently limited, the aim of this study was to investigate the susceptibility of Ureaplasma spp. and M. hominis to tetracyclines and fluoroquinolones in France. The susceptibility of 1014 clinical isolates obtained in Bordeaux University Hospital (Bordeaux, France) between 2010 and 2015 was evaluated using two commercial kits, S.I.R. Mycoplasma (Bio-Rad) from 1 January 2010 to 5 October 2012 and MYCOFAST RevolutioN kit (ELITech Group) from 6 October 2012 to 31 December 2015. The MICs of isolates designated as resistant were determined using the broth microdilution assay. Additionally, the tet(M) gene and fluoroquinolone resistance-associated mutations were identified. Among 831 Ureaplasma spp. isolates, the tetracycline, levofloxacin and moxifloxacin resistance rates were 7.5%, 1.2% and 0.1%, respectively. Among 183 M. hominis isolates, the resistance rates were 14.8%, 2.7% and 1.6% for tetracycline, levofloxacin and moxifloxacin, respectively. Over the 6 year period, no significant change in resistance to tetracycline or fluoroquinolones was observed. The tet(M) gene was found in all tetracycline-resistant isolates. All levofloxacin-resistant isolates harboured a mutation in the parC or parE genes. Isolates that were also resistant to moxifloxacin harboured an additional mutation in the gyrA gene. The MYCOFAST RevolutioN kit significantly overestimated levofloxacin and moxifloxacin resistance in Ureaplasma spp. isolates. Resistance to tetracycline and fluoroquinolones is limited in France in mycoplasmas but compared with a previous report in 1999-2002, a significant increase in tetracycline resistance among Ureaplasma spp. was observed. Ongoing monitoring of the antibiotic susceptibility of these urogenital mycoplasmas remains necessary.

Fluoroquinolone resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a Japanese tertiary hospital: silent shifting to CTX-M-15-producing K. pneumoniae.

Fluoroquinolone resistance (FQ-r) in extended-spectrum β-lactamase (ESBL) producers is an urgent health concern in countries where ESBL-producing K. pneumoniae (ESBL-Kpn) is prevalent. We investigated FQ-r in Japan where ESBL-Kpn is less prevalent. Clinical ESBL-Kpn isolates from 2011 to 2013 were collected in Nagasaki University Hospital. The ESBL genotypes included CTX-M-15, and the mechanisms of FQ-r through plasmid-mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining regions (QRDRs) were examined. Clonality was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multi-locus sequence typing was performed on selected isolates.Results/Key findings. Thirty ESBL-Kpn isolates, including seven levofloxacin-resistant isolates, were obtained from different patients. An increase in CTX-M-15-producing strains was observed during the study period (0/11 in 2011, 3/8 in 2012, and 5/11 in 2013). PMQR was detected in 53.3 % of the isolates and aac-(6')-Ib-cr was the most common (36.7 %). ST15 was observed in 60.0 % of the isolates, and for the most predominant ERIC-PCR profiles, 62.5 % of the isolates possessed the CTX-M-15 genotype and 71.4 % were levofloxacin-resistant. Levofloxacin-resistance was significantly more common in CTX-M-15 isolates (62.5 %) compared to non-CTX-M-15 isolates (9.1 %). Three QRDR mutations and aac(6')-Ib-cr, but not qnrB and qnrS, were significantly enriched in the CTX-M-15 isolates (100.0 %) compared to the non-CTX-M-15 isolates (13.6 %). Cumulatively, these results indicate that the epidemic strain, the CTX-M-15-producing K. pneumoniae ST15, is covertly spreading even when ESBL producers are not prevalent. Monitoring these epidemic strains and ESBLs in general is important for quickly identifying health crises and minimizing future risks from FQ-r ESBL-Kpn.

Multiclonal emergence of levofloxacin-resistant group B Streptococcus, Taiwan.

This study investigated the trend in antimicrobial resistance among group B Streptococcus (GBS) from a national surveillance programme in Taiwan and delineated characteristics of and factors associated with levofloxacin-resistant isolates. Clinical isolates of all sample types and patient groups were collected from multiple hospitals biennially between 2002 and 2012. Susceptibilities to different antibiotics were determined by broth microdilution. Molecular studies of levofloxacin-resistant isolates included serotyping, PFGE, mutations in the QRDRs and MLST. A total of 1559 isolates were tested and all remained susceptible to penicillin, cephalosporins, meropenem and vancomycin. However, levofloxacin resistance increased from 2.2% (range 0%-3.3%) in 2002-06 to 6.2% (5.9%-7.5%) in 2008-12 (P = 0.016). Among the 88 levofloxacin-resistant isolates, the majority (79.5%) had the GyrA(S81L)+ParC(S79F/Y) double mutations and most (54.5%) were also resistant to clindamycin, erythromycin and tetracycline. The predominant genotype of the levofloxacin-resistant isolates was ST19/serotype III (43.2%). Four previously unreported genotypes, ST1 and its single-locus variants (ST920 and ST922)/serotype VI (28.4%) and ST1/serotype II (18.2%), were found to have circulated locally. Serotype III isolates were predominately from urine and female genital tract specimens and <65-year-old adult outpatients, while serotype II and VI isolates were mostly from respiratory and urine samples and >65-year-old inpatients. Multivariate analysis revealed that elderly age and respiratory samples were independent factors associated with levofloxacin resistance. Multiclonal emergence and dissemination of levofloxacin-resistant GBS isolates occurred in healthcare and community settings in Taiwan. Continuous molecular-level surveillance is important to detect new epidemic trends.

Molecular Characterization of Italian Isolates of Fluoroquinolone-Resistant Streptococcus agalactiae and Relationships with Chloramphenicol Resistance.

A total number of 368 clinical isolates of Streptococcus agalactiae (group B Streptococcus, GBS) were collected in 2010-2016 from three hospitals in a region of central Italy. Fluoroquinolone (FQ)-resistant isolates were selected using levofloxacin. Levofloxacin-resistant (LR) strains (11/368, 2.99%) were characterized for several features, and their FQ resistance was analyzed phenotypically and genotypically using seven additional FQs. Their gyrA and parC quinolone resistance-determining regions were sequenced. Of the 11 LR isolates, 10 showed high-level and 1 low-level resistance. The former isolates exhibited higher minimal inhibitory concentrations also of the other FQs and all shared one amino acid substitution in ParC (Ser79Phe) and one in GyrA (Ser81Leu); only Ser79Phe in ParC was detected in the low-level LR isolate. The 11 LR strains exhibited distinctive relationships between their susceptibilities to non-FQ antibiotics and typing data. Remarkably, despite the very rare occurrence of chloramphenicol resistance in S. agalactiae, no <4 of the 11 LR isolates were chloramphenicol-resistant. Studies of GBS resistance to FQs in Europe remain scarce, notwithstanding the emergence of multidrug-resistant isolates. The incidence of LR GBS isolates is still limited in Italy, consistent with the moderate (though growing) rates reported in Europe, and much lower than the very high rates reported in East Asia. The intriguing relationships between FQ and chloramphenicol resistance deserve further investigation.

Molecular epidemiology and antimicrobial susceptibility of Clostridium difficile isolated from the Chinese People’s Liberation Army General Hospital in China

Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe responsible for antibiotic-associated diarrhoea. Its virulence is associated with the production of endotoxins A and B and endospores, which can cause symptoms, such as diarrhoea, toxic megacolon, and pseudomembranous colitis. Given the increasing elderly population and the well-recognized problem of over-prescribing of broad-spectrum antibiotics, it is critical to have an understanding of molecular epidemiology and antimicrobial susceptibility in China. This study analyzed the toxin types and multilocus sequence typing (MLST) results of 74 clinical isolates of C. difficile after the glutamate dehydrogenase (GDH) screening test and anaerobic culture. The minimum inhibitory concentrations (MICs) of four different antibiotics were determined for all of the isolates, and the bacterial resistance mechanisms were investigated. Sixty-five strains (75%) were toxigenic, including 54 tcdA-positive, tcdB-positive, and cdtA/cdtB-negative strains (A+B+CDT−) and nine A−B+CDT− strains. Eleven strains (14.9%) were non-toxigenic. All clinical isolates were classified into 26 MLST genotypes, with the predominant type being ST-54 (18.9%). All isolates were susceptible to vancomycin. The tetracycline, clindamycin, and levofloxacin resistance rates were 1.4%, 36.5%, and 20.3%, respectively. The expression of tet(M), erm(B), and mutations of gyrA and/or gyrB were observed in the tetracycline-, clindamycin-, and levofloxacin-resistant isolates, respectively.

Serotype Distribution and Antimicrobial Resistance of Streptococcus pneumoniae in the Philippines, 2004-2011

Objective. Scarcity of data on the serotype composition and antibiotic resistance of invasive pneumococci from developing countries has been noted. We describe in this study the serogroup distribution and antimicrobial resistance patterns of Streptococcus pneumoniae in the Philippines from 2004-2011. Methodology. S. pneumoniae isolated from patients with invasive pneumococcal disease were referred to the Antimicrobial Resistance Surveillance Reference Laboratory from 2004 to 2011. Typing of isolates was done through slide agglutination and antimicrobial susceptibility was determined following CLSI methods. Results. The penicillin-resistant meningitis isolates were of serotypes 1, 5 and 14 which are covered by PCV10 and 13. The erythromycin resistant isolates were serotype 9 while cotrimoxazole resistant isolates were serotypes 1, 5, 6, 12 and 14. Forty-one percent of the cotrimoxazole resistant isolates are covered by PCV7, and 88% are covered by both PCV10 and PCV13. Levofloxacin resistant isolates were of serotypes 5 and 23 with PCV7 coverage of 50% and PCV10 and PCV13 coverages of 100%. Conclusion. S. pneumoniae serotypes causing IPD in the country is largely similar to the dominant IPD serotypes worldwide. The serotype distribution in the Philippines remained stable from 2004 to 2011 and antimicrobial resistance among the isolates remained low. The serotypes of antibiotic resistant S. pneumoniae in this study were not similar with known serotype resistance profiles in other Asian countries. With the inclusion of PCV in the national free national immunization program of the country beginning 2013, continued surveillance of prevailing pneumococcal serotypes should be done to monitor any shift in the prevalence of PCV associated serotypes to guide disease control measures including control of emergence of resistant pneumococcal isolates.

Contribution of mexAB-oprM and mexXY (-oprA) efflux operons in antibiotic resistance of clinical Pseudomonas aeruginosa isolates in Tabriz, Iran

Overexpression of efflux pumps is one of the most important mechanisms that contributes to intrinsic and acquired resistance to antibiotics in Pseudomonas aeruginosa. The present study evaluated the role of MexAB-OprM and MexXY (-OprA) efflux pump overexpression in antibiotics resistance of P. aeruginosa clinical isolates. One-hundred clinical isolates of P. aeruginosa were obtained from four hospitals of Tabriz city in Northwest Iran. Isolates were identified and evaluated by the disk diffusion method and agar dilution in order to determine antibiotic resistance. Effect of Phenylalanine Arginine beta-Naphthylamide (PAβN) on susceptibility to various anti-Pseudomonas antimicrobials and expression levels of mexB and mexY using quantitative real-time PCR were determined in the clinical isolates. Random Amplified Polymorphic DNA Typing (RAPD-PCR) was used for genotyping of the isolates. The most and least effective antibiotics tested were colistin and ofloxacin, respectively. Seventy-one percent of the isolates were found as multidrug resistant (resistant to at least three different classes of antibiotics). Among ciprofloxacin and levofloxacin resistant isolates, 39.6% and 28.5% of them showed four-fold reduction in MIC with PAβN, respectively. Sixty-two percent and 65% of isolates overexpressed mexB and mexY, respectively. Sixty six isolates showed overexpression of both mexB and mexY efflux genes. Moreover, 76% and 88.7% of MDR isolates were mexB and mexY overexpressed, respectively. There were 30 different RAPD types in this study which were clustered into 6 clones. The study indicated that there is a significant correlation between the expression of efflux pumps and the resistance to most anti-pseudomonal antibiotics.

Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States

Our whole genome sequence (WGS) pipeline was assessed for accurate prediction of antimicrobial phenotypes. For 2316 invasive pneumococcal isolates recovered during 2015 we compared WGS pipeline data to broth dilution testing (BDT) for 18 antimicrobials. For 11 antimicrobials categorical discrepancies were assigned when WGS-predicted MICs and BDT MICs predicted different categorizations for susceptibility, intermediate resistance or resistance, ranging from 0.9% (tetracycline) to 2.9% (amoxicillin). For β-lactam antibiotics, the occurrence of at least four-fold differences in MIC ranged from 0.2% (meropenem) to 1.0% (penicillin), although phenotypic retesting resolved 25%–78% of these discrepancies. Non-susceptibility to penicillin, predicted by penicillin-binding protein types, was 2.7% (non-meningitis criteria) and 23.8% (meningitis criteria). Other common resistance determinants included mef (475 isolates), ermB (191 isolates), ermB + mef (48 isolates), tetM (261 isolates) and cat (51 isolates). Additional accessory resistance genes (tetS, tet32, aphA-3, sat4) were rarely detected (one to three isolates). Rare core genome mutations conferring erythromycin-resistance included a two-codon rplD insertion (rplD69-KG-70) and the 23S rRNA A2061G substitution (six isolates). Intermediate cotrimoxazole-resistance was associated with one or two codon insertions within folP (238 isolates) or the folA I100L substitution (38 isolates), whereas full cotrimoxazole-resistance was attributed to alterations in both genes (172 isolates). The two levofloxacin-resistant isolates contained parC and/or gyrA mutations. Of 11 remaining isolates with moderately elevated MICs to both ciprofloxacin and levofloxacin, seven contained parC or gyrA mutations. The two rifampin-resistant isolates contained rpoB mutations. WGS-based antimicrobial phenotype prediction was an informative alternative to BDT for invasive pneumococci.