Change In GyrA Research Articles

Resistance to Fluoroquinolones in Pseudomonas aeruginosa from Human, Animal, Food and Environmental Origin: The Role of CrpP and Mobilizable ICEs.

Fluoroquinolone resistance and the associated genetic mechanisms were assessed by antimicrobial susceptibility and whole genome sequencing in 56 Pseudomonas aeruginosa strains from human, animal, food and environmental origins. P. aeruginosa PAO1, PA7 and PA14 reference strains were also included in the study. Twenty-two strains (37%) were resistant to, at least, one fluoroquinolone agent. Correlation between the number of changes in GyrA and ParC proteins and the level of fluoroquinolone resistance was observed. Mutations or absence of genes, such as mexZ, mvaT and nalD encoding efflux pumps regulators, were also found in resistant strains. The crpP gene was detected in 43 strains (72.9%; 17 of them non-clinical strains), and coded seven different CrpP variants, including a novel one (CrpP-7). The crpP gene was located in 23 different chromosomal mobile integrative and conjugative elements (ICEs), inserted in two tRNAs integration sites. A great variety of structures was detected in the crpP-ICEs elements, e.g., the fimbriae related cup clusters, the mercury resistance mer operon, the pyocin S5 or S8 bacteriocin encoding genes, and mobilization genes. The location of crpP-like genes in mobilizable ICEs and linked to heavy metal resistance and virulence factors is of significant concern in P. aeruginosa. This work provides a genetic explanation of the fluoroquinolone resistance and crpP-associated pathogenesis of P. aeruginosa from a One-Health approach.

Clonal distribution and antimicrobial resistance of methicillin-susceptible and -resistant Staphylococcus aureus strains isolated from broiler farms, slaughterhouses, and retail chicken meat

Colonization of food-producing animals by antimicrobial-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), has become a serious public health problem worldwide. In the current study, clonal diversities of livestock-associated S. aureus isolates collected from broiler farms, slaughterhouses, and retail chicken meat were examined. Two-hundred S. aureus isolates (43 MRSA and 157 methicillin-susceptible S. aureus [MSSA] isolates) were analyzed to determine 1) the genotypes of the isolates (multilocus sequence, agr, and spa types), 2) the methicillin resistance phenotype and staphylococcal cassette chromosome mec (SCCmec) types, 3) the antimicrobial resistance profiles, and 4) the mutational changes in gyrA, gyrB, parC, and parE in fluoroquinolone-resistant isolates. Fifteen different sequence types (STs) of MSSA strains displaying a relatively high degree of genetic diversity were detected in broiler farms, slaughterhouses, and retail chicken meat. In contrast to MSSA, 2 dominant genetic lineages of MRSA (ST692-SCCmecV with t2249 spa type, and ST188-SCCmecIVa with spa type t189) were found in healthy broilers. The high prevalence of ST692 and ST188 in healthy broilers is associated with high levels of multiple antimicrobial-resistance phenotypes, particularly fluoroquinolone resistance. All fluoroquinolone-resistant isolates carried double point mutations in gyrA (S84L) and parC (S80F), regardless of STs or methicillin resistance. Notably, only the ST188 lineage carried an additional third mutation in gyrB (D494N), correlating with enhanced ciprofloxacin minimum inhibitory concentration values versus the strains with double mutations. These results provide important insights into the genetic diversity of antimicrobial-resistant S. aureus strains associated with the chicken meat production chain, including healthy broilers, in Korea.

Molecular Characterization of Fluoroquinolone-Resistant Bartonella bacilliformis.

The presence of amino acid changes in GyrA, GyrB, ParC, ParE, and in a proposed chromosomal chloramphenicol acetyl transferase (CAT), as well as mutations at 23S rRNA, were established by PCR and sequencing in 38 B. bacilliformis clinical isolates from four different areas in Peru. Eighteen out of 24 (75%) isolates showing ciprofloxacin resistance for both disk-diffusion and e-test presented amino acid substitutions in GyrA (G89C, six isolates, A91V, 1 isolate) GyrB (S474F, 10 isolates) or both (GyrA D95N and GyrB S474F, one isolate). Two out of 14 susceptible isolates presented amino acid substitutions at GyrB (S474F) or a double substitution GyrA D95N and GyrB S474F. Of note, ciprofloxacin-resistant isolates were recovered in the four areas studied. No amino acid change was observed at ParC or ParE. Only one isolate showed chloramphenicol resistance, but no alteration was present in either 23S rRNA or CAT. B. bacilliformis resistant to quinolones are extended throughout Peru, with amino acid substitutions at GyrA or GyrB as the main, albeit not exclusive, cause. B. bacilliformis seems to have an apparent facility to develop mutations on GyrB outside the classical positions 91, 95 of GyrA and 85, 88 of ParC.

Emergence of Invasive Serotype Ib Sequence Type 10 Group B Streptococcus Disease in Chinese Infants Is Driven by a Tetracycline-Sensitive Clone

BackgroundGroup B streptococcus (GBS) is a leading cause of serious infections in infants. The extensive use of tetracycline has led to the selection of specific resistant and infectious GBS clones. The sequence type (ST) 10 GBS strain, causing invasive infections in infants, is becoming prevalent in China. We aimed to understand the clinical and microbiological characteristics of this GBS strain.MethodsWe conducted a retrospective study on infants with invasive GBS disease from the largest women’s and children’s medical center in Shanxi and collected data between January 2017 and October 2020. GBS isolates were analyzed by capsule serotyping, genotyping, antibiotic resistance, and surface protein genes.ResultsAll ST10 isolates belonged to serotype Ib; type Ib/ST10 strains were responsible for 66.7% (14/21, P < 0.05) of infant invasive GBS infections during the period and all resulted in late-onset (LOD) and late LOD disease (14/14). Infants with type Ib/ST10 GBS disease had significantly higher rates of meningitis (9/14, 64.3%, p < 0.05) and clinical complications (5/14, 35.7%, p < 0.05). The Ib/ST10 GBS isolates had limited genetic diversity, clustered in the CC10/bca/PI-1 + PI-2a genetic lineage, showed resistance to erythromycin, lincomycin, and fluoroquinolones and sensitivity to tetracycline, and possessed genes ermT, ermB, and amino acid changes in gyrA and parC.ConclusionsThe probable clonal expansion can result in severe infections in infants and ongoing emergence of multi-drug resistant isolates. Continued monitoring for type Ib/ST10 GBS infections is warranted.

Presence and Transfer of Antimicrobial Resistance Determinants in Escherichia coli in Pigs, Pork, and Humans in Thailand and Lao PDR Border Provinces.

This study aimed to investigate antimicrobial resistance (AMR) characteristics of Escherichia coli isolates from pig origin (including pigs, pig carcass, and pork) and humans in Thailand and Lao People's Democratic Republic (PDR) border provinces. The majority of the E. coli isolates from Thailand (69.7%) and Lao PDR (63.3%) exhibited multidrug resistance. Class 1 integrons with resistance gene cassettes were common (n = 43), of which the most predominant resistance gene cassette was aadA1. The percentage of extended-spectrum beta-lactamase (ESBL) producers was 3.4 in Thailand and 3.2 in Lao PDR. The ESBL genes found were blaCTX-M14, blaCTX-M27, and blaCTX-M55, of which blaCTX-M55 was the most common (58.6%). Ser-83-Leu and Asp-87-Asn were the predominant amino acid changes in GyrA of ciprofloxacin-resistant isolates. Twenty-two percent of all isolates were positive for qnrS. Class 1 integrons carrying aadA1 from pigs (n = 1) and ESBL genes (blaCTX-M55 and blaCTX-M14) from pigs (n = 2), pork (n = 1), and humans (n = 7) were located on conjugative plasmids. Most plasmids (29.3%) were typed in the IncFrepB group. In conclusion, AMR E. coli are common in pig origin and humans in these areas. The findings confirm AMR as One Health issue, and highlight the need for comprehensive and unified collaborations within and between sectors on research and policy.

The determination of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in carbapenem resistant Klebsiella pneumonia ST11 and ST76 strains isolated from patients in Heilongjiang Province, China

BackgroundThere is increasing resistance to carbapenems among Klebsiella pneumoniae,and fluoroquinolones (FQ) are increasingly used to treat infections from extended-spectrum β- lactamase(ESBLs) and carbapenemase-producing Klebsiella pneumoniae. However, the acquisition of plasmid-mediated quinolone resistance (PMQR) or the spontaneous mutation of the quinolone resistance-determining regions (QRDR) of the gyrA and parC genes can severely affect the therapeutic effect of quinolones. The goal of this study was to investigate the molecular determinants of FQ resistance(FQ-R) in carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from Heilongjiang Province,China. Materials and methodsWe isolated 40 strains of CRKP from a treatment center in the eastern part of Heilongjiang Province from January 2016 to December 2018. The VITEK2 Compact analyzer was used to identify and detect drug sensitivity. Different types of drug resistance genes were detected by polymerase chain reaction (PCR). PCR and DNA sequencing were used to assess the presence of qnrA, qnrB, qnrS,qepA and acc(6′) Ib-cr genes,which are plasmid-encode genes that can contribute to resistance. The sequences of gyrA and parC genes were sequenced and compared with the sequences of standard strains to determine if mutations were present.Multi-site sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were performed on the strains to assess homology. ResultsThe isolated CRKP strains showed rates of resistance to fluoroquinolones of 22.5% to 42.5%. The resistance rate of ciprofloxacin was significantly higher than that of levofloxacin.Nine CRKP strains (22.5%) showed co-resistance to ciprofloxacin and levofloxacin.The quinolone resistant strains were screened for plasmid-encoded genes that can contribute to resistance (PMQR genes).Among the 17 quinolone resistant strains,one strain contained no PMQR genes,twelve strains contained two PMQR genes,and four strains contained four PMQR genes.Acc (6′) Ib-cr was the most frequently detected PMQR gene, detected in 95% of strains tested (38 of 40) and in 94.1% of the quinolone-resistant strains (16 of 17). The qepA gene encoding an efflux pump was not detected in any strains.No isolate carried five different PMQRs simultaneously.Changes of S83I and D87G changes in gyrA, and the S80I change in parC,which were mediated by QRDR,were identified in two isolates,which showed resistance to both ciprofloxacin and levofloxacin.Most of the FQ-R strains(58.8%,10/17) belong to ST(sequence type) 76, which is dominant in the local area, while all the mutant strains (100%,2/2),that differ in at least one site from standard bacteria, belong to the ST11 group. The strains were isolated from a hospital where there had been a recent outbreak of ST76 type CRKP in the neurosurgery ward and intensive care unit. ConclusionCRKP strains were identified that were insensitive or even resistant to quinolones,and this resistance is common in Heilongjiang Province of eastern China;fluoroquinolone-resistance in these clinical CRKP strains is a complex interplay between PMQR determinants and mutations in gyrA and parC.The resistance level caused by QRDR mutation is higher than that caused by PMQR, however, the high frequency of PMQR genes in the isolated CRKP strains suggests the potential for impact of these genes.PMQR determinants are often found in carbapenemase-producing or ESBLs-producing Klebsiella pneumoniae,and some resistance genes,such as:SHV,TEM, CTX-M-15,and OXA-1 are closely associated with FQ-R. Finally, geographical factors can affect the emergence and spread of PMQR and QRDR.Some genetic lineages have higher potential risks, and continuous close monitoring is required.

Nationwide Stepwise Emergence and Evolution of Multidrug-Resistant Campylobacter jejuni Sequence Type 5136, United Kingdom.

We examined whole-genome–sequenced Campylobacter jejuni and C. coli from 2012–2015 isolated from birds and human stool samples in North East Scotland for the presence of antimicrobial resistance genes. We found that sequence type (ST) 5136 (clonal complex 464) was the most prevalent multidrug-resistant strain of C. jejuni exclusively associated with poultry host reservoirs and recovered from human cases of campylobacteriosis. Tetracycline resistance in ST5136 isolates was due to a tet(O/32/O) mosaic gene, ampicillin resistance was conferred by G → T transversion in the −10 promoter region of blaOXA-193, fluoroquinolone resistance was due to C257T change in gyrA, and aminoglycoside resistance was conferred by aac. Whole-genome analysis showed that the strain ST5136 evolved from ST464. The nationwide emergence of ST5136 was probably due to stepwise acquisition of antimicrobial resistance genes selected by high use of β-lactam, tetracycline, fluoroquinolone, and aminoglycoside classes of drugs in the poultry industry.

Targeting DNA Gyrase to Combat Mycobacterium tuberculosis: An Update.

DNA gyrase is a clinically validated drug target, currently targeted only by fluoroquinolone class of antibacterials. However, owing to increasing drug resistance as well as a concomitant reduction in the availability of newer classes of antibiotics, fluoroquinolones are increasingly being over-utilized in order to treat serious infections, including multi-drug resistant tuberculosis. This, in turn, increases the probability of resistance to fluoroquinolones, which is mediated by a single amino acid change in gyrA, leading to class-wide resistance. In this review, we provide an overview of the recent progress in identifying novel scaffolds which target DNA gyrase and provide an update on their discovery and development status.

Prevalence and mechanism of fluoroquinolone resistance in clinical isolates of Proteus mirabilis in Japan

Fluoroquinolone (FQ) and cephalosporin (CEP) resistance among Enterobacteriaceae has been increasingly reported. FQ resistance occurs primarily through mutations in DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). CEP resistance in Enterobacteriaceae is mainly due to the production of CTX-M type extended-spectrum β-lactamases. Although prevalence and mechanisms of FQ and CEP resistance in Enterobacteriaceae such as Escherichia coli have been well studied, little is known about Proteus mirabilis in Japan. In this study, we assessed the prevalence and mechanism of FQ resistance in Japanese clinical isolates of P. mirabilis. We collected 5845 P. mirabilis isolates from eight hospitals between 2000 and 2013. Prevalence of FQ resistance was calculated as the annual average percentage of all P. mirabilis isolates. We selected 50 isolates exhibiting susceptibility, intermediate resistance, or resistance to levofloxacin (LVX) and identified amino acid substitutions in GyrA, GyrB, ParC, and ParE. The prevalence of FQ-resistant P. mirabilis gradually increased from 2001 to 2004, reaching 16.6% in 2005, and has remained relatively high (13.3–17.5%) since then. Low-level LVX-resistant strains (MIC, 8–16 mg/L) showed significant changes in GyrB (S464Y or -I, or E466D). High-level LVX-resistant strains (MIC, 32–128 mg/L) displayed significant changes in GyrA (E87K) and ParE (D420N). The highest-level LVX-resistant strains (MIC, ≥ 256 mg/L) presented significant changes in GyrA (E87K or -G), GyrB (S464I or -F), and ParE (D420N). Our findings suggest that substitutions in GyrA (E87) and ParE (D420) have played an important role in the emergence of high-level LVX-resistant P. mirabilis isolates (MIC, ≥ 32 mg/L) in Japan.

Emerging high-level ciprofloxacin resistance and molecular basis of resistance in Salmonella enterica from humans, food and animals

Disease caused by nontyphoidal serotypes of Salmonella enterica is the leading cause of foodborne illness worldwide. Many serotypes have also developed resistance to antimicrobials commonly used for the treatment of salmonellosis. Here we analyze 179 ciprofloxacin-resistant isolates identified among 3680 Salmonella isolated from humans, food, animals, and water collected in Shanghai, China. They were assessed for antimicrobial susceptibility, plasmid-mediated quinolone resistance (PMQR) determinants, and mutations in quinolone resistance determination regions (QRDRs); genetic relatedness was examined using PFGE. Ciprofloxacin resistance in Salmonella increased from 2.3% in 2006 to 5.9% in 2012. Multidrug resistance was common, and most carried mutations in QRDR (97.2%) and PMQR determinants (71.5%). Mutations frequently included changes in gyrA: Ser83Phe (53.6%) and Asp87Asn (35.8%), and in parC: Thr57Ser (53.1%) and Ser80Arg (44.1%). Mutations in parC and parE without changes in gyrA were identified in S. Derby and most S. Thompson. Among PMQR determinants, aac(6′)-Ib-cr (62.0%) and oqxA/oqxB (33.5%/33.0%) were most common, and conferred resistance without target mutations in five S. Typhimurium isolates. PFGE analysis revealed that S. Typhimurium, isolated from pork and aquatic products, and S. Indiana, isolated from chicken, were highly similar to isolates from humans, suggesting these products be the major source of ciprofloxacin-resistant infections. Our findings highlight the important role QRDRs and PMQR play in ciprofloxacin resistance of Salmonella, and reveal the potential sources of the pathogen associated with human infections.

GyrA and/or ParC alterations of Haemophilus influenzae strains isolated from the urethra of men with acute urethritis.

Of 73 clinical strains of Haemophilus influenzae isolated from the urethra of men with urogenital infections, we enrolled 6 strains (8.2%) with levofloxacin (LVFX) minimum inhibitory concentrations (MICs) of ≥0.03μg/ml in this study. All the strains were isolated from non-gonococcal urethritis (NGU). We amplified the quinolone resistance-determining region of the gyrA gene and the analogous region of the parC gene from bacterial DNAs by PCR and sequenced the PCR products. Two strains with a LVFX MIC of 0.03μg/ml had an amino acid change of Asp88 to Gly in GyrA. One with a LVFX MIC of 0.06μg/ml had a change of Asp88 to Tyr in GyrA. Two with respective LVFX MICs of 0.12 and 0.25μg/ml had a change of Ser84 to Leu in GyrA. One with a LVFX MIC of 1μg/ml had changes of Ser84 to Leu in GyrA and of Ser84 to Ile in ParC. Multilocus sequence typing showed two strains with a change of Asp88 to Gly in GyrA had the same sequence type, but the others had sequence types different from each other. Single amino acid changes in GyrA alone or single changes in both GyrA and ParC could contribute to decreased susceptibility to fluoroquinolones in H.influenzae isolates from NGU. Most of the isolates with GyrA and/or ParC alterations would be multiclonal. The prevalence of such isolates would be relatively low, and they would still be susceptible to fluoroquinolones commonly prescribed for treatment of NGU.

Resistance mechanisms against quinolones in Mycoplasma capricolum subsp. capricolum

Quinolones interact with bacterial DNA gyrase and topoisomerase IV, the subunits of which are encoded by gyrA/gyrB and parC/parE, respectively. The aim of this study was to evaluate the relationship between changes in these genes and quinolone susceptibility of Mycoplasma capricolum subsp. capricolum (Mcc). Using in vitro selected resistant mutants and field isolates from goats, predicted amino acid changes in gyrA, gyrB and parC were associated with higher minimum inhibitory concentration values for quinolones. Alterations in parC predicted amino acid sequences were most frequently associated with quinolone resistance in Mcc.

Resistance gene pool to co-trimoxazole in non-susceptible Nocardia strains.

The soil-borne pathogen Nocardia sp. causes severe cutaneous, pulmonary, and central nervous system infections. Against them, co-trimoxazole (SXT) constitutes the mainstay of antimicrobial therapy. However, some Nocardia strains show resistance to SXT, but the underlying genetic basis is unknown. We investigated the presence of genetic resistance determinants and class 1–3 integrons in 76 SXT-resistant Nocardia strains by PCR and sequencing. By E test, these clinical strains showed SXT minimum inhibitory concentrations of ≥32:608 mg/L (ratio of 1:19 for trimethoprim: sulfamethoxazole). They belonged to 12 species, being the main representatives Nocardia farcinica (32%), followed by N. flavorosea (6.5%), N. nova (11.8%), N. carnea (10.5%), N. transvalensis (10.5%), and Nocardia sp. (6.5%). The prevalence of resistance genes in the SXT-resistant strains was as follows: sul1 and sul2 93.4 and 78.9%, respectively, dfrA(S1) 14.7%, blaTEM-1 and blaZ 2.6 and 2.6%, respectively, VIM-2 1.3%, aph(3′)-IIIa 40.8%, ermA, ermB, mefA, and msrD 2.6, 77.6, 14.4, and 5.2%, respectively, and tet(O), tet(M), and tet(L) 48.6, 25.0, and 3.9%, respectively. Detected amino acid changes in GyrA were not related to fluoroquinolone resistance, but probably linked to species polymorphism. Class 1 and 3 integrons were found in 93.42 and 56.57% strains, respectively. Class 2 integrons and sul3 genes were not detected. Other mechanisms, different than dfrA(S1), dfrD, dfrF, dfrG, and dfrK, could explain the strong trimethoprim resistance shown by the other 64 strains. For first time, resistance determinants commonly found in clinically important bacteria were detected in Nocardia sp. sul1, sul2, erm(B), and tet(O) were the most prevalent in the SXT-resistant strains. The similarity in their resistome could be due to a common genetic platform, in which these determinants are co-transferred.

Molecular characterization of fluoroquinolone resistance in nontypeable Haemophilus influenzae clinical isolates.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of respiratory infections in adults, who are frequently treated with fluoroquinolones. The aims of this study were to characterize the genotypes of fluoroquinolone-resistant NTHi isolates and their mechanisms of resistance. Among 7,267 H. influenzae isolates collected from adult patients from 2000 to 2013, 28 (0.39%) were ciprofloxacin resistant according to Clinical and Laboratory Standards Institute (CLSI) criteria. In addition, a nalidixic acid screening during 2010 to 2013 detected five (0.23%) isolates that were ciprofloxacin susceptible but nalidixic acid resistant. Sequencing of their quinolone resistance-determining regions and genotyping by pulse-field gel electrophoresis and multilocus sequence typing of the 25 ciprofloxacin-resistant isolates available and all 5 nalidixic acid-resistant isolates were performed. In the NTHi isolates studied, two mutations producing changes in two GyrA residues (Ser84, Asp88) and/or two ParC residues (Ser84, Glu88) were associated with increased fluoroquinolone MICs. Strains with one or two mutations (n = 15) had ciprofloxacin and levofloxacin MICs of 0.12 to 2 μg/ml, while those with three or more mutations (n = 15) had MICs of 4 to 16 μg/ml. Long persistence of fluoroquinolone-resistant strains was observed in three chronic obstructive pulmonary disease patients. High genetic diversity was observed among fluoroquinolone-resistant NTHi isolates. Although fluoroquinolones are commonly used to treat respiratory infections, the proportion of resistant NTHi isolates remains low. The nalidixic acid disk test is useful for detecting the first changes in GyrA or in GyrA plus ParC among fluoroquinolone-susceptible strains that are at a potential risk for the development of resistance under selective pressure by fluoroquinolone treatment.

Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

In a previous report (T. J. Dougherty, A. Nayar, J. V. Newman, S. Hopkins, G. G. Stone, M. Johnstone, A. B. Shapiro, M. Cronin, F. Reck, and D. E. Ehmann, Antimicrob Agents Chemother 58:2657-2664, 2014), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4- to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16- and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Streptococcus parauberis의 퀴놀론 내성 증가와 Topoisomerase 유전자에서의 돌연변이 신속 분석

To investigate the acquisition of quinolone resistance, we examined mutations in the quinolone resistance-determining region (QRDR) of type II topoisomerase genes in ciprofloxacin (CIP)-resistant clinical isolates and in vitro mutants of Streptococcus parauberis. The CIP-resistant clinical isolates had one base change responsible for a Ser-79<TEX>${\rightarrow}$</TEX>Thr in the QRDR of parC. However, the CIP-resistant in vitro mutants had an altered QRDR of parC (Ser-79<TEX>${\rightarrow}$</TEX>Ile) that differed from that of the isolates. None of the CIP-resistant S. parauberis clinical isolates or in vitro mutants exhibited amino acid changes in gyrA or gyrB. However, even though involvement in the increased resistance was not clear, an Arg-449<TEX>${\rightarrow}$</TEX>Ser mutation outside of the QRDR of parE was detected in CIP-resistant mutant 2P1. These results suggest that the topoisomerase IV gene, parC (and possibly parE, as well), is the primary ciprofloxacin target in S. parauberis. Additionally we established a high-resolution melting (HRM) assay capable of detecting the dominant mutation in four type II topoisomerase genes conferring ciprofloxacin resistance. These rapid and reliable assays may provide a convenient method of surveillance for genetic mutations conferring antibiotic resistance.

Emergence of quinolone-resistant, topoisomerase-mutant Brucella after treatment with fluoroquinolones in a macrophage experimental infection model

Aim of the studyTo determine the activity of fluoroquinolones (FQ) and the selection of FQ-resistant mutants in a macrophage experimental infection model (MEIM). Material and methodsCanine macrophages were inoculated with Brucella melitensis ATCC 23457 (WT), achieving intracellular counts of around 105CFU/mL. Cell cultures were incubated in the presence of ciprofloxacin (CIP), levofloxacin (LEV), moxifloxacin (MOX), and doxycycline (DOX). After cell lysis, surviving microorganisms were plated for count purposes, and plated onto antibiotics-containing media for mutant selection. Topoisomerases mutations were detected by PCR and sequencing. ResultsBacterial counts after cell lysis were 14.3% (CIP), 65.3% (LEV), and 75% (MOX) lower compared to the control. Quinolone-resistant mutants emerged in cell cultures containing CIP and LEV with a frequency of around 0.5×10−3. All mutants showed an Ala87Val change in GyrA. Mutants had FQs MICs around 10×WT. The ability of these mutants for infecting new macrophages and the intracellular lysis after antibiotic exposure did not change significantly. No 2nd step FQ-resistant mutants were selected from 1st step mutants. ConclusionsIntracellular activity of FQs is low against WT and gyrA-mutant Brucella. FQs easily select gyrA mutants in MEIM. The ability of mutants for infecting new macrophages remains unchanged. In this MEIM, 2nd step mutants do not emerge.

Fluoroquinolone-Resistant Pneumococci: Dynamics of Serotypes and Clones in Spain in 2012 Compared with Those from 2002 and 2006

In Spain, rates of ciprofloxacin resistance in pneumococci were low during the last decade (2.6% in 2002 and 2.3% in 2006). In 2012, the rate remained at 2.3%, equivalent to 83 of 3,621 isolates. Of the 83 resistant isolates, 15 showed a low level (MIC of 4 to 8 μg/ml) and 68 a high level (MIC of 16 to 128 μg/ml) of ciprofloxacin resistance. Thirteen low-level-resistant isolates had single changes in ParC, one had a single ParE change, and one did not present any mutations. High-level-resistant isolates had GyrA changes plus additional ParC and/or ParE changes: 51, 15, and 2 isolates had 2, 3, or 4 mutations, respectively. Although 24 different serotypes were observed, 6 serotypes accounted for 51.8% of ciprofloxacin-resistant isolates: 8 (14.5%), 19A (10.8%), 11A (7.2%), 23A (7.2%), 15A (6.0%), and 6B (6.0%). A decrease in pneumococcal 7-valent conjugate vaccine (PCV7) serotypes was observed from 2006 (35.7%) to 2012 (16.9%), especially of serotype 14 (from 16.3% to 2.4%; P<0.001). In comparison with findings in 2006, multidrug resistance was greater in 2012 (P=0.296), mainly due to the increased presence and/or emergence of clonal complexes associated with non-PCV7 serotypes: CC63 expressing serotypes 8, 15A, and 19A; CC320 (with serotype 19A); and CC42 (with serotype 23A). Although rates of ciprofloxacin resistance remained low and stable throughout the last decade, changes in serotype and genotype distributions were observed in 2012, notably the expansion of a preexisting multidrug-resistant clone, CC63, and the emergence of the CC156 clone expressing serotype 11A.

Clonal spread of Klebsiella pneumoniae producing OXA-1 betalactamase in a Spanish hospital.

Multi-drug resistant Klebsiella pneumoniae isolates are associated with nosocomial infections, in which colonized patients act as a reservoir and source of cross-infection for other patients. In this study, the antimicrobial susceptibility of K. pneumoniae was tested by microdilution using the commercial method MicroScan (Siemens). The genetic relatedness of K. pneumoniae strains was determined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PCR experiments were carried out to obtain primer sets and positive PCR products were purified and sequenced. From May 2007 until December 2009, 98 clonally related K. pneumoniae isolates were detected from clinical samples of 38 patients admitted to the University Hospital of Bellvitge, Barcelona, Spain, including 27 admitted to the intensive care unit (ICU). The most important sources of the isolates were: lower respiratory tract (n = 12), urine (n = 12), and blood (n = 11). The strains were resistant to amoxicillin/clavulanic acid, piperacillin/tazobactam, tobramycin, amikacin, and ciprofloxacin, and had diminished susceptibility to cefepime. All the isolates shared a common PFGE pattern related to sequence type 14 after MLST analysis. In K. pneumoniae isolates and their transconjugants, the bla(OXA-1) gene was located in the variable region of a class I integron that also contains the aac(6')Ib-cr gene. Sequencing of the quinolone resistance determinant regions of gyrA and parC revealed a S83F change in GyrA and no changes in ParC.

Novel Conserved Genotypes Correspond to Antibiotic Resistance Phenotypes of E. coli Clinical Isolates

Current efforts to understand antibiotic resistance on the whole genome scale tend to focus on known genes even as high throughput sequencing strategies uncover novel mechanisms. To identify genomic variations associated with antibiotic resistance, we employed a modified genome-wide association study; we sequenced genomic DNA from pools of E. coli clinical isolates with similar antibiotic resistance phenotypes using SOLiD technology to uncover single nucleotide polymorphisms (SNPs) unanimously conserved in each pool. The multidrug-resistant pools were genotypically similar to SMS-3-5, a previously sequenced multidrug-resistant isolate from a polluted environment. The similarity was evenly spread across the entire genome and not limited to plasmid or pathogenicity island loci. Among the pools of clinical isolates, genomic variation was concentrated adjacent to previously reported inversion and duplication differences between the SMS-3-5 isolate and the drug-susceptible laboratory strain, DH10B. SNPs that result in non-synonymous changes in gyrA (encoding the well-known S83L allele associated with fluoroquinolone resistance), mutM, ligB, and recG were unanimously conserved in every fluoroquinolone-resistant pool. Alleles of the latter three genes are tightly linked among most sequenced E. coli genomes, and had not been implicated in antibiotic resistance previously. The changes in these genes map to amino acid positions in alpha helices that are involved in DNA binding. Plasmid-encoded complementation of null strains with either allelic variant of mutM or ligB resulted in variable responses to ultraviolet light or hydrogen peroxide treatment as markers of induced DNA damage, indicating their importance in DNA metabolism and revealing a potential mechanism for fluoroquinolone resistance. Our approach uncovered evidence that additional DNA binding enzymes may contribute to fluoroquinolone resistance and further implicate environmental bacteria as a reservoir for antibiotic resistance.