Decreased Fluoroquinolone Susceptibility Research Articles

Nontyphoidal Salmonella Invasive Disease: Challenges and Solutions.

Nontyphoidal Salmonella are a leading cause of community-onset bacteremia and other serious infections in sub-Saharan African countries where large studies indicate that they are an uncommon cause of moderate-to-severe diarrhea. Approximately 535 000 nontyphoidal Salmonella invasive disease illnesses and 77 500 deaths were estimated to occur in 2017; 422 000 (78.9%) illnesses and 66 500 (85.9%) deaths in countries in sub-Saharan Africa. Lineages of Salmonella enterica serovar Typhimurium sequence type (ST) 313 and lineages of Salmonella enterica serovar Enteritidis ST11 dominate as causes of invasive disease. A major reservoir for these specific strains outside of humans has not been identified to date. Human fecal shedding of such strains is common in areas where nontyphoidal Salmonella invasive disease incidence is high. The case-fatality ratio of nontyphoidal Salmonella invasive disease is approximately 15%. Early diagnosis and treatment are needed to avert fatal outcomes. Antimicrobial resistance, including multiple drug resistance, decreased fluoroquinolone susceptibility, and resistance to third-generation cephalosporins, is increasing in prevalence and is likely to further compromise patient outcomes. Naturally acquired immunity against invasive disease develops in children aged >3 years in endemic areas, likely mediated in part by the sequential acquisition of T-cell immunity, followed by antigen-specific immunoglobulin G antibodies. Vaccines in preclinical or clinical development include live-attenuated S. enterica serovar Typhimurium, nontyphoidal S. enterica core and O-polysaccharide glycoconjugates, multiple antigen-presenting system complexes, and generalized modules for membrane antigens vaccines. The latter are in phase I trials in Europe and Africa. Both vaccine use, and other effective, evidence-based nonvaccine interventions, are needed to prevent and control nontyphoidal Salmonella invasive disease.

Rapid fluoroquinolone resistance detection in Pseudomonas aeruginosa using mismatch amplification mutation assay-based real-time PCR.

Background. Antimicrobial resistance (AMR) is an ever-increasing global health concern. One crucial facet in tackling the AMR epidemic is earlier and more accurate AMR diagnosis, particularly in the dangerous and highly multi-drug-resistant ESKAPE pathogen, Pseudomonas aeruginosa.Objectives. We aimed to develop two SYBR Green-based mismatch amplification mutation assays (SYBR-MAMAs) targeting GyrA T83I (gyrA248) and GyrA D87N, D87Y and D87H (gyrA259). Together, these variants cause the majority of fluoroquinolone (FQ) AMR in P. aeruginosa.Methods. Following assay validation, the gyrA248 and gyrA259 SYBR-MAMAs were tested on 84 Australian clinical P. aeruginosa isolates, 46 of which demonstrated intermediate/full ciprofloxacin resistance according to antimicrobial susceptibility testing.Results. Our two SYBR-MAMAs correctly predicted an AMR phenotype in the majority (83%) of isolates with intermediate/full FQ resistance. All FQ-sensitive strains were predicted to have a sensitive phenotype. Whole-genome sequencing confirmed 100 % concordance with SYBR-MAMA genotypes.Conclusions. Our GyrA SYBR-MAMAs provide a rapid and cost-effective method for same-day identification of FQ AMR in P. aeruginosa. An additional SYBR-MAMA targeting the GyrB S466Y/S466F variants would increase FQ AMR prediction to 91 %. Clinical implementation of our assays will permit more timely treatment alterations in cases where decreased FQ susceptibility is identified, leading to improved patient outcomes and antimicrobial stewardship.

Surveillance of Fluoroquinolone Resistance in Wisconsin: Geographic Variation and Impact of Revised CLSI Breakpoints

Objective: Many clinical microbiology laboratories procure antimicrobial susceptibility testing data using guidelines established by Clinical and Laboratory Standards Institute (CLSI). When necessary, CLSI revises interpretive breakpoints in efforts to improve clinical correlation, with two revisions relative to fluoroquinolone agents occurring in 2019. The purpose of this investigation was to determine the impact of fluoroquinolone breakpoint revisions on Wisconsin clinical isolates of Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa. Design: Multi-center laboratory surveillance, with testing at a single location utilizing standardized media and susceptibility testing protocols.Methods: From the Surveillance of Wisconsin Organisms for Trends in Antimicrobial Resistance and Epidemiology (SWOTARE) program, levofloxacin and ciprofloxacin minimum inhibitory concentration (MIC) values for 1911, 1521, and 1463 Wisconsin isolates of E. coli, P. mirabilis, and P. aeruginosa, respectively, were determined by broth microdilution testing. In separate data analyses, all MIC data were interpreted using CLSI breakpoints published prior to 2019, then secondarily by using CLSI breakpoints published since 2019 (which reflect lower breakpoints for both levofloxacin and ciprofloxacin resistance). Findings were further stratified by Wisconsin Department of Health Services region.Results: Up to 3.2% decreased statewide fluoroquinolone susceptibility was observed for E. coli isolates, while 5.1% and 6.3% decreases in levofloxacin susceptibility were noted for P. aeruginosa and P. mirabilis isolates, respectively, when revised breakpoints were applied. E. coli isolates from the Western region and P. mirabilis isolates from the Southeastern region demonstrated significant shifts toward decreased fluoroquinolone susceptibility upon application of revised breakpoints. Northern region P. mirabilis isolates exhibited consistently decreased fluoroquinolone susceptibility.Conclusions: Fluoroquinolone resistance has been underreported in Wisconsin as a whole, yet geographic variability continues to exist. Targeted annual surveillance is important to identify and monitor resistance trending. Compilations of SWOTARE surveillance data can be utilized to predict the impact of future CLSI interpretive breakpoint revisions in Wisconsin.

Genetic diversity of Salmonella Paratyphi A isolated from enteric fever patients in Bangladesh from 2008 to 2018.

BackgroundThe proportion of enteric fever cases caused by Salmonella Paratyphi A is increasing and may increase further as we begin to introduce typhoid conjugate vaccines (TCVs). While numerous epidemiological and genomic studies have been conducted for S. Typhi, there are limited data describing the genomic epidemiology of S. Paratyphi A in especially in endemic settings, such as Bangladesh.Principal findingsWe conducted whole genome sequencing (WGS) of 67 S. Paratyphi A isolated between 2008 and 2018 from eight enteric disease surveillance sites across Bangladesh. We performed a detailed phylogenetic analysis of these sequence data incorporating sequences from 242 previously sequenced S. Paratyphi A isolates from a global collection and provided evidence of lineage migration from neighboring countries in South Asia. The data revealed that the majority of the Bangladeshi S. Paratyphi A isolates belonged to the dominant global lineage A (67.2%), while the remainder were either lineage C (19.4%) or F (13.4%). The population structure was relatively homogenous across the country as we did not find any significant lineage distributions between study sites inside or outside Dhaka. Our genomic data showed presence of single point mutations in gyrA gene either at codon 83 or 87 associated with decreased fluoroquinolone susceptibility in all Bangladeshi S. Paratyphi A isolates. Notably, we identified the pHCM2- like cryptic plasmid which was highly similar to S. Typhi plasmids circulating in Bangladesh and has not been previously identified in S. Paratyphi A organisms.SignificanceThis study demonstrates the utility of WGS to monitor the ongoing evolution of this emerging enteric pathogen. Novel insights into the genetic structure of S. Paratyphi A will aid the understanding of both regional and global circulation patterns of this emerging pathogen and provide a framework for future genomic surveillance studies.

Development of molecular biological tools for the rapid determination of antibiotic susceptibility of Mycoplasma hyopneumoniae isolates

Mycoplasma hyopneumoniae is the etiologic agent of porcine enzootic pneumonia, a contagious respiratory disease, causing significant economic losses worldwide. Antibiotic treatment is commonly utilised in the pig industry to control M. hyopneumoniae infection. Since the conventional antibiotic susceptibility test is time-consuming, taking up to weeks’ period, antibiotics are usually empirically chosen.Certain single nucleotide polymorphisms in the parC (C239A/T, G250A) and gyrA (G242C, C247 T, A260 G) genes show correlation with decreased fluoroquinolone susceptibility by the change of the target site. Furthermore, the nucleotide alteration A2059 G in the 23S rRNA sequence correlates with significantly decreased macrolide and lincosamide susceptibility of M. hyopneumoniae. Mismatch amplification mutation assays (MAMA) and high resolution melt (HRM) analysis, capable to detect the mentioned resistance markers, were developed in the present study, in order to provide susceptibility data in a considerably shorter time than the conventional methods. The results of the MAMA and HRM assays were congruent with the results of the conventional antibiotic susceptibility method of the tested M. hyopneumoniae field isolates. The sensitivity of the MAMAs was 103-104 copy numbers, while that of the HRM assay was 105-106 copy numbers.To the best of our knowledge this was the first time that MAMA and HRM assays were developed for the rapid detection of decreased fluoroquinolone, macrolide or lincosamide susceptibility in M. hyopneumoniae strains.

Nalidixic acid screening test in detection of decreased fluoroquinolone susceptibility in Salmonella typhi isolated from blood.

To determine the validity of nalidixic acid screening test in the detection of high MICs of fluoroquinolone against Salmonella(S.) typhi isolated from blood and correlate zone diameters of ofloxacin with that of MIC value for nalidixic acid sensitive and resistant strains. Cross-sectional analytical study. Clinical Microbiology Laboratory of the Aga Khan Hospital, Karachi from January 2002 to December 2003. Two hundred S. typhi isolates from blood were included for nalidixic acid screening and ofloxacin susceptibility. Antibiotic susceptibilities for both the antibiotics were obtained by disc diffusion method whereas MICs were determined by standard agar dilution method as recommended by NCCLS guidelines. Sensitivity, specificity and correlation between both antimicrobial susceptibility methods were calculated and results expressed as scattergrams. The results broadly classify S. typhi isolates into nalidixic acid resistant strains with no zone of inhibition around 30 mug nalidixic acid disc and nalidixic acid sensitive strains with mean zone of inhibition of 24.9 mm. All S. typhi isolates with ofloxacin MIC of capital ZHE, Cyrillic 0.125 microg/ml were found to be nalidixic acid resistant (MIC capital ZHE, Cyrillic32 microg/ml) whereas the isolates with ofloxacin MIC 0.06 microg/ml were nalidixic acid sensitive (MIC 8 microg/ml). Screening for nalidixic acid resistance was found to be 100% sensitive and 97% specific in identifying S. typhi strains with reduced susceptibility to fluoroquinolone (MIC capital ZHE, Cyrillic 0.125 microg/ml). Nalidixic acid resistance as a screening method is proved to be significant in identifying S. typhi isolates with reduced susceptibility to fluoroquinolones. It is also suggested that inhibition zone of 25 mm around 5 microg ofloxacin disc is appropriate as a selection criterion to detect S. typhi isolates with reduced susceptibility to fluoroquinolones.

Case Series of Imported Enteric Fever at a Referral Center in Tokyo, Japan: Antibiotic Susceptibility and Risk Factors for Relapse.

Owing to the increase in Salmonella strains with decreased fluoroquinolone susceptibility in the endemic areas, we have been treating enteric fever with intravenous ceftriaxone empirically since 2007. In this study, we reevaluated our treatment protocol. This retrospective cohort study was conducted at a single institute in Tokyo, Japan, between January 2006 and December 2013. Enteric fever was defined as isolation of Salmonella Typhi or Salmonella Paratyphi A, B, and C from the blood and/or stool of patients with fever. Of the 35 patients with imported enteric fever, 28 (80%) had returned from south Asia. Ciprofloxacin-susceptible strains were detected in only 12% of the cases. The isolates showed excellent susceptibility to ampicillin (91%), chloramphenicol (94%), ceftriaxone (97%), and azithromycin (97%). One case of Salmonella Paratyphi B was excluded, and of the remaining 34 patients, 56% were treated with ceftriaxone alone, 26% with ceftriaxone then fluoroquinolone, and 9% with levofloxacin alone. The overall relapse rate was 6.1%; however, among those receiving ceftriaxone monotherapy, the relapse rate was 11% (N = 2). The relapse group was characterized by longer times to treatment initiation (P = 0.035) and defervescence (> 7 days) after treatment initiation (P = 0.022). In such cases, we recommend that ceftriaxone treatment be continued for > 4 days after defervescence or be changed to fluoroquinolone if the strains are found to be susceptible to prevent relapse. Furthermore, ampicillin and chloramphenicol, which are no longer prescribed, may be reconsidered as treatment options in Asia.

Description and plasmid characterization of the qnrD determinant in Proteeae in Wenzhou, Southern China.

Only limited information is available about the detailed characteristics of qnrD, a plasmid-mediated quinolone resistance (PMQR) gene. This study aimed to understand the distribution of qnrD and the characterization of qnrD-carrying plasmids in Proteeae. The distribution of qnrD genes was investigated by polymerase chain reaction (PCR) amplification in 203 consecutive nonduplicate clinical isolates of Proteeae collected from inpatients at the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China. The minimum inhibitory concentrations (MICs) of antibiotics were measured by agar dilution method and other PMQR determinants were also determined by PCR. qnrD was positioned via Southern hybridization and the transferability of qnrD-carrying plasmids was achieved by conjugation experiment. The genetic environment of qnrD was investigated by sequencing, and chromosomal polymorphism for qnrD-positive strains was analyzed by pulsed-field gel electrophoresis (PFGE). Forty strains carried qnrD, showing decreased fluoroquinolone susceptibility or low-level fluoroquinolone resistance. qnrD was encoded on the plasmid of about 2.7kb or 5.2kb in length, which cannot be transferred by liquid conjugation or filter mating, but can be successfully transferred by transduction. The transformants showed 62.5-300-fold increases in the MICs of quinolones compared with the recipient. The plasmids carrying qnrD showed a high similarity with that of Providencia spp. and Proteus vulgaris. PFGE analysis demonstrated that these isolates were divergent and not clone related. qnrD could have originated from Proteeae or presented in these bacteria as a reservoir; furthermore, qnrD could be transferred and spread within the same or across different bacterial species if the plasmids acquired mobile elements under antimicrobial selective pressures.

Epidemiology, Clinical Presentation, Laboratory Diagnosis, Antimicrobial Resistance, and Antimicrobial Management of Invasive Salmonella Infections.

Salmonella enterica infections are common causes of bloodstream infection in low-resource areas, where they may be difficult to distinguish from other febrile illnesses and may be associated with a high case fatality ratio. Microbiologic culture of blood or bone marrow remains the mainstay of laboratory diagnosis. Antimicrobial resistance has emerged in Salmonella enterica, initially to the traditional first-line drugs chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole. Decreased fluoroquinolone susceptibility and then fluoroquinolone resistance have developed in association with chromosomal mutations in the quinolone resistance-determining region of genes encoding DNA gyrase and topoisomerase IV and also by plasmid-mediated resistance mechanisms. Resistance to extended-spectrum cephalosporins has occurred more often in nontyphoidal than in typhoidal Salmonella strains. Azithromycin is effective for the management of uncomplicated typhoid fever and may serve as an alternative oral drug in areas where fluoroquinolone resistance is common. In 2013, CLSI lowered the ciprofloxacin susceptibility breakpoints to account for accumulating clinical, microbiologic, and pharmacokinetic-pharmacodynamic data suggesting that revision was needed for contemporary invasive Salmonella infections. Newly established CLSI guidelines for azithromycin and Salmonella enterica serovar Typhi were published in CLSI document M100 in 2015.

Description of an original integron encompassing blaVIM-2, qnrVC1 and genes encoding bacterial group II intron proteins in Pseudomonas aeruginosa.

A burn unit of a hospital in Tunis underwent an endemic situation caused by imipenem-resistant Pseudomonas aeruginosa. For nine non-repetitive isolates of a clonal VIM-2-producing strain, the blaVIM-2 genetic background was characterized and the associated qnrVC1 gene molecularly analysed. The imipenem resistance mechanism was investigated by phenotypic and molecular tests, and resistance transfer was studied by conjugation and transformation experiments. The integron's structure was characterized by sequencing, and qnrVC1 expression was explored after cloning experiments. The nine VIM-2-producing strains were collected from eight patients and one environmental sample. All transfer assays failed, suggesting a chromosomal location of blaVIM-2. This latter was found to be part of a class 1 integron of ∼7500 bp, which also contains blaOXA-2, aadA1 and two copies of the aadB, arr-6 and qnrVC1 genes. qnrVC1 exhibited higher homology with the chromosomally encoded qnr genes of Vibrionaceae than with plasmid-mediated qnr genes of Enterobacteriaceae. The qnrVC1 gene cassette possesses a promoter allowing its expression, and it conferred decreased fluoroquinolone susceptibility to Escherichia coli. Additionally, on the same integron, genes encoding an uncommon group IIC-attC intron were detected. A VIM-2-producing P. aeruginosa outbreak led us to characterize an integron harbouring a qnrVC1 cassette and a new group IIC-attC intron. This is the first known description of a qnr determinant in a P. aeruginosa strain. Its presence conferred a low level of resistance to quinolones in E. coli, which might favour the emergence of highly resistant mutants.

Structure based in silico analysis of quinolone resistance in clinical isolates of Salmonella Typhi from India.

Enteric fever is a major cause of morbidity in several parts of the Indian subcontinent. The treatment for typhoid fever majorly includes the fluoroquinolone group of antibiotics. Excessive and indiscriminate use of these antibiotics has led to development of acquired resistance in the causative organism Salmonella Typhi. The resistance towards fluoroquinolones is associated with mutations in the target gene of DNA Gyrase. We have estimated the Minimum Inhibitory Concentration (MIC) of commonly used fluoroquinolone representatives from three generations, ciprofloxacin, ofloxacin, levofloxacin and moxifloxacin, for 100 clinical isolates of Salmonella Typhi from patients in the Indian subcontinent. The MICs have been found to be in the range of 0.032 to 8 μg/ml. The gene encoding DNA Gyrase was subsequently sequenced and point mutations were observed in DNA Gyrase in the quinolone resistance determining region comprising Ser83Phe/Tyr and Asp87Tyr/Gly. The binding ability of these four fluoroquinolones in the quinolone binding pocket of wild type as well as mutant DNA Gyrase was computationally analyzed by molecular docking to assess their differential binding behaviour. This study has revealed that mutations in DNA Gyrase alter the characteristics of the binding pocket resulting in the loss of crucial molecular interactions and consequently decrease the binding affinity of fluoroquinolones with the target protein. The present study assists in understanding the underlying molecular and structural mechanism for decreased fluoroquinolone susceptibility in clinical isolates as a consequence of mutations in DNA Gyrase.

English

CONTEXT: Enteric fever prevails to be a considerable health issue worldwide. Ciprofloxacin became the mainstay of its therapy after multidrug resistant salmonellae emerged. Treatment failure was soon recorded with ciprofloxacin, the index being in-vitro resistance to nalidixic acid. Chloramphenicol sensitive strains have resurfaced. Resistance to alternatives- macrolides is cropping up though not documented widely. These variations in addition to the fact that India is witnessing escalating resistance to antibiotics, stress the significance of continual surveillance of salmonellae resistance. INTENT: The study undertaken intends to document the current deviations in the antibiogram of the Salmonella isolates from blood culture, with a perspective of evaluating ciprofloxacin susceptibility and macrolide resistance, further considering, if there is a need to modify the antibiotic strategies for this condition. METHODOLOGY: The isolates from blood cultures from January 2012 to September 2013 obtained using commercial BACTEC 9120 system, sub-cultured and processed for determining salmonellae serotypes were further subjected to drug susceptibility testing for patterning their resistance. MICs to ciprofloxacin of nalidixic acid resistant (NAR) isolates were recognized. RESULTS: Salmonella was isolated from 114 out of 8210 blood culture specimens received. There were 74 Salmonella Typhi isolates, 39 Salmonella Paratyphi A and 1 Salmonella Paratyphi B. Highest resistance of salmonellae (85.96%) was exhibited to nalidixic acid and highest susceptibility (100%) to ceftriaxone. Decreased ciprofloxacin susceptibility-DCS (MIC of 0.125-1μg/ml) was noticed in around 90% of NAR salmonellae. Isolates demonstrated sizable resistance (42.10%) to azithromycin. Only three strains were multidrug resistant. INTERPRETATION AND CONCLUSION: Routine screening of nalidixic acid sensitivity should be encouraged as a means to predict decreased fluoroquinolone susceptibility, thereby preventing therapeutic failure. In order to avert the profound impact of antimicrobial resistance and to define the best possible therapy, periodic monitoring of Salmonella antibiogram is of prime value. The upsurge of occurrence of ciprofloxacin resistance, impending resistance to macrolide- azithromycin, re-emergence of chloramphenicol sensitive strains is of particular concern and necessitates a change towards 'evidence-based' treatment and rationalization of existing empirical therapy of enteric fever shifting the vital focus towards antibiotic recycling.

Antimicrobial Resistance in Invasive Non-typhoid Salmonella from the Democratic Republic of the Congo: Emergence of Decreased Fluoroquinolone Susceptibility and Extended-spectrum Beta Lactamases

BackgroundCo-resistance against the first-line antibiotics ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole or multidrug resistance (MDR) is common in non typhoid Salmonella (NTS). Use of alternative antibiotics, such as fluoroquinolones or third generation cephalosporins is threatened by increasing resistance, but remains poorly documented in Central-Africa.Methodology/Principal findingsAs part of a microbiological surveillance study in DR Congo, blood cultures were collected between 2007 and 2011. Isolated NTS were assessed for serotype and antimicrobial resistance including decreased ciprofloxacin susceptibility and extended-spectrum beta-lactamase (ESBL) production. In total, 233 NTS isolates (representing 23.6% of clinically significant organisms) were collected, mainly consisting of Salmonella Typhimurium (79%) and Salmonella Enteritidis (18%). The majority of NTS were isolated in the rainy season, and recovered from children ≤2 years old. MDR, decreased ciprofloxacin susceptibility, azithromycin and cefotaxime resistance were 80.7%, 4.3%, 3.0% and 2.1% respectively. ESBL production was noted in three (1.3%) isolates. Decreased ciprofloxacin susceptibility was associated with mutations in codon 87 of the gyrA gene, while ESBLs all belonged to the SHV-2a type.Conclusions/SignificancePresence of almost full MDR among NTS isolates from blood cultures in Central Africa was confirmed. Resistance to fluoroquinolones, azithromycin and third generation cephalosporins is still low, but emerging. Increased microbiological surveillance in DR Congo is crucial for adapted antibiotic therapy and the development of treatment guidelines.

Nalidixic Acid Resistance and Clonal Expansion of <i>Salmonella enterica </i>Serotype Paratyphi a Isolates in Yuxi City, China

The aim of our study was to understand the nalidixic acid resistance and clonal expansion of Salmonella enterica serotype Paratyphi A (SPA) recovered from patients with paratyphoid fever A in Yuxi City from 1999 to 2008. A typing method is needed to determine the epidemic clones of the isolates. Antimicrobial susceptibility testing was performed with 4 060 SPA isolates. 186 isolates were typed by pulsed-field gel electrophoresis (PFGE) with SpeI, and the MICs for nalidixic acid, ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, and gatifloxacin were determined. The incidence of resistance to nalidixic acid of the isolates increased from 12.5% in 1999, 82.2% in 2000, 93.0% in 2001, to100% in 2008. Nalidixic acid-susceptible (NAS) isolates predominated in 1999 but was replaced by nalidixic acid-resistant (NAR) isolates after 2000. The frequencies of intermediary and susceptibility (Kirby-Bauer disc diffusion technique) to ciprofloxacin were 17.0% and 83.0% before 2005, 65.3% and 34.7% in 2008, respectively. A group of 166 NAR and 20 NAS isolates of SPA were randomly selected. SpeI digestion of 186 isolates gave nine different PFGE patterns with predominance of the SpeI01 and SpeI02 epidemic patterns. All NAR isolates studied had a reduced susceptibility to fluoroquinolone, the MICs were higher than that for 20 NAS isolates.We validate the use of the nalidixic acid screening test in the detection of decreased fluoroquinolone susceptibility in SPA. PFGE patterns SpeI01 and SpeI02, the main clones of the epidemics, are highly prevalent and NAR isolates in Yuxi. Recommendation for the emergency treatments of outbreaks and epidemics caused by a fully fluoroquinolone-resistant strain can be made. PFGE with SpeI is a useful technique to differentiate SPA.

A multiplex single nucleotide polymorphism typing assay for detecting mutations that result in decreased fluoroquinolone susceptibility in Salmonella enterica serovars Typhi and Paratyphi A

ObjectivesDecreased susceptibility to fluoroquinolones has become a major problem for the successful therapy of human infections caused by Salmonella enterica, especially the life-threatening typhoid and paratyphoid fevers.MethodsBy using Luminex xTAG beads, we developed a rapid, reliable and cost-effective multiplexed genotyping assay for simultaneously detecting 11 mutations in gyrA, gyrB and parE of S. enterica serovars Typhi and Paratyphi A that result in nalidixic acid resistance (NalR) and/or decreased susceptibility to fluoroquinolones.ResultsThis assay yielded unambiguous single nucleotide polymorphism calls on extracted DNA from 292 isolates of Salmonella Typhi (NalR = 223 and NalS = 69) and 106 isolates of Salmonella Paratyphi A (NalR = 24 and NalS = 82). All of the 247 NalR Salmonella Typhi and Salmonella Paratyphi A isolates were found to harbour at least one of the target mutations, with GyrA Phe-83 as the most common one (143/223 for Salmonella Typhi and 18/24 for Salmonella Paratyphi A). We also identified three GyrB mutations in eight NalS Salmonella Typhi isolates (six for GyrB Phe-464, one for GyrB Leu-465 and one for GyrB Asp-466), and mutations GyrB Phe-464 and GyrB Asp-466 seem to be related to the decreased ciprofloxacin susceptibility phenotype in Salmonella Typhi. This assay can also be used directly on boiled single colonies.ConclusionsThe assay presented here would be useful for clinical and reference laboratories to rapidly screen quinolone-resistant isolates of Salmonella Typhi and Salmonella Paratyphi A, and decipher the underlying genetic changes for epidemiological purposes.

Decreased fluoroquinolone susceptibility in mutants of Salmonella serovars other than Typhimurium: detection of novel mutations involved in modulated expression of ramA and soxS

Mutants of five Salmonella enterica serovars were investigated for structural changes in regulatory regions known to be involved in the up-regulation of efflux pumps. Five Salmonella field isolates and mutants, in which efflux pump inhibitor tests previously pointed towards an up-regulation of efflux, plus one negative control were included in the study. MIC values were determined of antibiotics that were indicative of AcrAB overexpression. The regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of original strains and mutants were sequenced and compared. The gene expression of acrA, tolC, ramA and soxS was assessed by quantitative real-time PCR. Conjugation experiments and tet gene PCRs were performed to explain unexpected variations in MIC values of tetracycline. In four mutant strains, changes in the ramRA regulatory region, causing up-regulation of ramA, were detected. These changes comprised point mutations and deletions of 10 or 15 bp within the ramR gene and a single bp exchange located in the binding site of the RamR protein in Salmonella Infantis, Paratyphi and Livingstone mutants. An insertion of 49 bp within the soxR gene was involved in soxS up-regulation and enhanced efflux activity in the fifth mutant from Salmonella Virchow. The loss of tetracycline resistance in one Salmonella Paratyphi mutant could be explained by the loss of a plasmid carrying a tet(A) gene. Changes in the ramR-ramA region as well as in the soxR gene occur in mutants of Salmonella serovars other than Typhimurium and seem to be involved in the up-regulation of efflux activity.

Epidemiological shift, seasonal variation and antimicrobial susceptibility patterns among enteric fever pathogens in south India

Between January 2005 and December 2006, a higher incidence of paratyphoid fever (53.8%) compared to typhoid fever (44.9%) has been observed at a tertiary hospital in South India. A definite seasonal pattern of incidence is seen in paratyphoid fever (peak incidence during October-December, i.e., post monsoon period) but not in typhoid fever. Decreased fluoroquinolone susceptibility is much higher in S. Paratyphi A (98.8%) as compared to S. Typhi (46.5%). These findings are of importance in therapeutic decision making, development of vaccination strategies and implementing public health measures for disease control.

Novel Variant of the qnrB Gene, qnrB12 , in Citrobacter werkmanii

Three epidemiologically and clonally unrelated Citrobacter werkmanii isolates of poultry origin from Germany (Table [1][1]) were investigated for the mechanisms responsible for decreased fluoroquinolone susceptibility. The MICs, determined by broth macrodilution ([10][2]), varied between 8 and >256

Molecular mechanisms of decreased susceptibility to fluoroquinolones in avian Salmonella serovars and their mutants selected during the determination of mutant prevention concentrations

Salmonella enterica isolates of six serovars and mutants obtained during determination of mutant prevention concentrations (MPCs) were investigated for mechanisms of decreased susceptibility to fluoroquinolones. The quinolone resistance determining regions (QRDRs) of gyrA, gyrB, parC and parE genes were sequenced. MIC values were determined in the presence/absence of the efflux pump inhibitors carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) or Phe-Arg-beta-naphthylamide (PA beta N). PCR assays for the quinolone resistance genes qnrA, qnrB, qnrS or aac(6')-Ib-cr were applied. The MPC values of ciprofloxacin (MPC(CIP)) were determined for all isolates and selected mutants were investigated for their quinolone resistance mechanisms. In contrast to 11 nalidixic acid-susceptible isolates, 24 nalidixic acid-resistant isolates exhibited single mutations in gyrA (Asp-87 --> Tyr, Gly, Asn or Ser-83 --> Phe, Tyr) or parC (Thr-57 --> Ser). While CCCP had no influence on the MICs, PA beta N decreased the MIC(CIP) values by 1-3 dilution steps and MIC(NAL) values by up to 6 dilution steps. Of the resistance genes investigated, only qnrS was present, in a single Salmonella Infantis isolate. The MPC(CIP) values were 4-64-fold higher than the MICs and ranged between 1-16 and 0.12-1 mg/L, respectively, for isolates resistant or susceptible to nalidixic acid. Only mutants obtained from formerly nalidixic acid-susceptible isolates developed single mutations in gyrA or gyrB. In field isolates and mutants, target site mutations and efflux seem to be important mechanisms for decreased fluoroquinolone susceptibility. Mutants derived during MPC determination from field isolates already harbouring single-step mutations in gyrA did not exhibit further mutations in any target genes.

Fluoroquinolone treatment of experimental Salmonella enterica serovar Typhimurium DT104 infections in chickens selects for both gyrA mutations and changes in efflux pump gene expression

To determine the efficacy of enrofloxacin (Baytril) in chickens in eradicating three different resistance phenotypes of Salmonella enterica and to examine the resistance mechanisms of resulting mutants. In two separate replicate experiments (I and II), three strains of Salmonella enterica serovar Typhimurium DT104 [strain A, fully antibiotic-sensitive strain; strain B, isogenic multiple antibiotic-resistant (MAR) derivative of A; strain C, veterinary penta-resistant phenotype strain containing GyrA Phe-83], were inoculated into day-old chicks at approximately 10(3) cfu/bird. At day 10, groups of chicks (n =10) were given either enrofloxacin at 50 ppm in their drinking water for 5 days or water alone (control). Caecal contents were monitored for presence of Salmonella and colonies were replica plated to media containing antibiotics or overlaid with cyclohexane to determine the proportion of isolates with reduced susceptibility. The MICs of antibiotics and cyclohexane tolerance were determined for selected isolates from the chicks. Mutations in topoisomerase genes were examined by DHPLC and expression of marA, soxS, acrB, acrD and acrF by RT-PCR. In experiment I, but not II, enrofloxacin significantly reduced the numbers of strain A compared with the untreated control group. In experiment II, but not I, enrofloxacin significantly reduced the numbers of strain B. Shedding of strain C was unaffected by enrofloxacin treatment. Birds infected with strains A and B gave rise to isolates with decreased fluoroquinolone susceptibility. Isolates derived from strain A or B requiring >128 mg/L nalidixic acid for inhibition contained GyrA Asn-82 or Phe-83. Isolates inhibited by 16 mg/L nalidixic acid were also less susceptible to antibiotics of other chemical classes and became cyclohexane-tolerant (e.g. MAR). These studies demonstrate that recommended enrofloxacin treatment of chicks rapidly selects for strains with reduced fluoroquinolone susceptibility from fully sensitive and MAR strains. It can also select for MAR isolates.