Detection of novel gyrA mutations in nalidixic acid-resistant isolates of Salmonella enterica from patients with diarrhoea

The aim of the current study was to detect mutations in the gyrA gene of quinolone-resistant Salmonella spp. isolates recovered in Tehran, Iran. Between April 2008 and September 2009, 174 Salmonella spp. were collected and assayed for quinolone resistance and detection of gyrA mutations. Isolates identified as Salmonella enterica were tested for susceptibility by the disk diffusion method. Polymerase chain reaction (PCR) amplification and sequencing of the gyrA gene segment encoding the quinolone resistance-determining region (QRDR) were performed for the nalidixic acid-resistant isolates. Amongst the 174 recovered Salmonella spp. isolates, 89 were resistant to nalidixic acid, of which 9 were resistant to enrofloxacin; 10 isolates had reduced susceptibility to nalidixic acid. None of the isolates were resistant to ciprofloxacin, but a single isolate showed reduced susceptibility. Twelve types of amino acid replacement were found in the QRDR region of GyrA, namely the previously described substitutions in positions 83 and 87 as well as five new substitutions Leu41-Pro, Arg47-Ser, Ser111-Thr, Ala118-Thr and Asp147-Gly. Double substitutions in both positions 83 and 87 were not identified. A Gly133-Glu substitution was identified in a single S. enterica serotype Typhi isolate.     

Predictive value of nalidixic acid resistance for detecting salmonellae with decreased ciprofloxacin susceptibility

Seventy-three Salmonella isolates classified as ciprofloxacin susceptible when using the criteria of the National Committee for Clinical Laboratory Standards were studied for nalidixic acid (NA) resistance. The aim of the study was to determine the predictive value of nalidixic acid resistance in screening for decreased ciprofloxacin susceptibility. We observed that isolates with decreased ciprofloxacin susceptibility were all resistant to nalidixic acid. Identification of nalidixic acid resistance by the disk diffusion method provided 100% sensitivity and a specificity of 98.4% in strains with minimum inhibitory concentrations (MICs) >0.008 mg/l.     

Comparison of Etest, agar dilution, broth microdilution and disk diffusion methods for testing in vitro activity of levofloxacin against Staphylococcus spp. isolated from neutropenic cancer patients

The susceptibility to levofloxacin of 194 consecutive staphylococcal (45 Staphylococcus aureus and 149 coagulase-negative staphylococci) isolates from neutropenic patients was determined by Etest and the results compared with those obtained using NCCLS-methods (broth microdilution, agar dilution and disk diffusion). Overall agreement at +/- 1log(2) dilution for Etest compared with broth microdilution and agar dilution was 99.0 and 83.5%, respectively. The Etest category agreement with broth microdilution and disk diffusion was 95.9 and 89.7%, respectively. Comparison of categories with Etest and agar dilution method gave only 67.0% absolute categorical agreement, with 29.9% minor errors and 10.7% major errors. No very major errors occurred by the four methods tested. Our results show that Etest is a valid alternative to the reference NCCLS-methods for monitoring the clinical usefulness of levofloxacin against staphylococci isolates from neutropenic patients.     

A comparison of antibiotic susceptibility testing methods for cotrimoxazole with Burkholderia pseudomallei

Melioidosis is caused by the Gram-negative soil saprophyte, Burkholderia pseudomallei and is endemic in tropical and subtropical regions of southeast Asia and northern Australia. Cotrimoxazole has been traditionally used for the therapy of melioidosis despite results indicating resistance often produced in the disc diffusion test against B. pseudomallei. This inconsistency was addressed by comparing this method with the agar dilution, MicroScan and E-test methods. The results demonstrated that by disc diffusion, 41.3% of 80 B. pseudomallei clinical isolates tested were susceptible to cotrimoxazole, whereas the MicroScan, agar dilution and the E-test demonstrated 92.5, 90 and 97.5% of the isolates to be susceptible, respectively. These results indicate that an MIC based method is required to test the susceptibility of B. pseudomallei against cotrimoxazole.     

Antimicrobial susceptibility and serogroups of Salmonella isolates from Riyadh, Saudi Arabia

The antimicrobial susceptibility and serogroups of 153 Salmonella strains isolated during a period of 22 months from both children and adults at a major teaching hospital in Riyadh, Saudi Arabia were studied. Antimicrobial susceptibility testing by comparative disc method and MIC determination by E-test method were performed on selected antimicrobial agents. For nalidixic acid and trimethoprim only the comparative disc method was used. Discrepancy between the two methods were noticed only in 1.3% of isolates. The majority of isolates from children (41%) were serogroup B, while those from adults (43%) were serogroup C1. The overall resistance was 16% to ampicillin and ampicillin/sulbactam, 13% to nalidixic acid, and 11% to chloramphenicol and trimethoprim/sulphamethoxazole. The resistance of Salmonella isolates to the so-called first line anti-Salmonella agents, i.e. ampicillin, chloramphenicol and trimethoprim/sulphamethoxazole, has increased compared to that reported 4 years ago from this Institution. Almost all isolates were susceptible to the second, and third generation cephalosporins, fluoroquinolones, aztreonam, mecillinam and gentamicin. Multiple drug resistance to two or more drugs was noticed in 16% of isolates, most of which were serogroup B. The majority of these multiple drug resistant isolates (96%) were ampicillin resistant and β-lactamase producers. Although these isolates showed reduced MICs to ampicillin/sulbactam, their MICs were still higher than the susceptibility breakpoint for this combination. The nalidixic acid-resistant isolates showed higher MICs to the fluoroquinolones compared to the nalidixic acid-sensitive isolates. Isolates from children showed higher resistance to some of the antimicrobial agents compared to those from adults.     

Comparative in vitro activity of enoxacin and other fluoroquinolones against multi-resistant strains of Salmonella typhi.

The in vitro activities of enoxacin, lomefloxacin, norfloxacin, ofloxacin, and pefloxacin against 274 strains of Salmonella typhi isolated from suspected typhoid fever patients (137 multi-resistant strains and 137 strains sensitive to chloramphenicol, ampicillin and/or co-trimoxazole) were determined using disk diffusion and agar dilution techniques. In vitro, enoxacin was active against all tested strains with a MIC90 and inhibition zone size against multi-resistant strains of 0.12 mg/l and 34 mm diameter, respectively. Similar results were found with the other fluoroquinolones. Enoxacin and other fluoroquinolones may be the therapy of choice in cases of typhoid fever caused by organisms resistant to the standard therapy, chloramphenicol.     

Prevalence and antimicrobial susceptibility patterns among gastroenteritis-causing pathogens recovered in Europe and Latin America and Salmonella isolates recovered from bloodstream infections in North America and Latin America: report from the SENTRY Antimicrobial Surveillance Program (2003)

Gastroenteritis-causing pathogens are the second leading cause of morbidity and mortality worldwide. Complicating the clinical diarrhoea syndrome is the emergence of antimicrobial resistance among the responsible bacterial pathogens. The reported increases in fluoroquinolone resistance in Salmonella, Shigella and Campylobacter have been extremely worrisome considering the primary role of ciprofloxacin as a treatment. In this study, 1479 bacterial isolates from gastroenteritis infections were collected in Europe and Latin America, which included Salmonella spp. (834; 56%), Shigella spp. (311; 21%), Campylobacter spp. (182; 12%) and Aeromonas spp. (72; 5%). The fluoroquinolones displayed the greatest activity against these pathogens, with only three non-Campylobacter spp. strains being non-susceptible using current Clinical and Laboratory Standards Institute (CLSI) breakpoint criteria. Whilst ciprofloxacin resistance in European and Latin American Salmonella was only 0.2% and 0.0%, respectively, a total of 16.2% and 12.9% of isolates were resistant to nalidixic acid, indicating possible first-step gyrA mutations. Among confirmed extended-spectrum beta-lactamase-producing Salmonella strains, CTX-M genes were detected in 15 originating from Russia. Erythromycin and azithromycin were the most potent agents tested against Campylobacter spp. (values of minimum inhibitory concentration for 90% of the organisms, 0.5 mg/L and 0.12 mg/L, respectively), with erythromycin displaying the highest susceptibility (91.1%). Salmonella isolates from bloodstream infections displayed antibiograms that were nearly identical to strains causing gastroenteritis. Considering the role that antimicrobial therapy plays in the management of moderate to severe bacterial gastroenteritis, global surveillance and local/national public health programmes can provide critical data illuminating the dissemination of resistance and guidance for empirical therapy.     

In vitro activity of tigecycline (GAR-936) and other antimicrobials against tetracycline- and ciprofloxacin-resistant Campylobacter clinical isolates

During 2003 in our hospital, 236 clinical faecal Campylobacter spp. were isolated, of which 166 (70%) were resistant to tetracycline and 199 (84%) were resistant to ciprofloxacin by the disk diffusion method. Resistance to both antimicrobial agents was found in 146 isolates (62%). The in vitro activity of tigecycline was compared with that of erythromycin, clindamycin and amoxicillin/clavulanic acid using the agar dilution method against 116 selected Campylobacter spp. that were resistant to both tetracycline and ciprofloxacin. The minimum inhibitory concentration at which 90% of the isolates were inhibited (MIC90) was 0.06 mg/L for tigecycline and 4, 2 and 1 microg/mL for amoxicillin/clavulanic acid, erythromycin and clindamycin, respectively. The high in vitro activity of tigecycline against tetracycline- and ciprofloxacin-resistant strains suggests a potential therapeutic role in the treatment of infections that involve Campylobacter spp.     

The antibacterial activity of fluoroquinolones against gram-negative bacteria isolated from Gifu Prefecture (2005)

We investigated the susceptibility to 6 fluoroquinolones against 433 strains of Gram-negative bacteria isolated from 6 medical facilities in Gifu prefecture between January and September in 2005, determined by the agar dilution methods in according with the Japan Society of Chemotherapy. We also investigated the correlation between the degree of resistance to fluoroquinolones and the amino acid substitutions in the quinolone resistance-determining region (QRDR). The tested clinical isolates were as follows, Salmonella spp.; 17 strains, Escherichia coli; 112 strains Citrobacter freundii; 35 strains, Enterobacter cloacae; 31 strains, Klebsiella pneumoniae; 73 strains, Proteus spp.; 18 strains, Providencia spp.; 3 strains, Morganella morganii; 14 strains, Serratia marcescens; 27 strains and Pseudomonas aeruginosa; 103 strains. The number of the strains resistant to ciprofloxacin (CPFX) (MIC > or = 6.25 microg/mL) was twenty (E. coli; 14 strains, E. cloacae; I strain, Proteus spp.; 2 strains and P. aeruginosa; 3 strains). Among these strains, 12 strain (E. coli; 11 strains and E. cloacae; 1 strain) were highly resistant to CPFX (MIC > or =25 microg/mL). The E. coli strains highly resistant to CPFX had the multiple amino acid mutations in QRDR of ParC an GyrA. However in other strains, there was no strains possessing multiple mutations in both ParC and GyrA.     

Comparative in vitro activity of enoxacin and other fluoro-quinolones against multi-resistant strains of Salmonella typhi

Summary

The in vitro activities of enoxacin, lomefloxacin, norfloxacin, ofloxacin, and pefloxacin against 274 strains of Salmonella typhi isolated from suspected typhoid fever patients (137 multi-resistant strains and 137 strains sensitive to chlor-amphenicol, ampicillin and/or co-trimoxazole) were determined using disk diffusion and agar dilution techniques. In vitro, enoxacin was active against all tested strains with a MIC₉₀ and inhibition zone size against multi-resistant strains of 0.12?mg/l and 34?mm diameter, respectively. Similar results were found with the other fluoroquinolones. Enoxacin and other fluoroquinolones may be the therapy of choice in cases of typhoid fever caused by organisms resistant to the standard therapy, chloramphenicol.     

Characterisation of Salmonella spp. mutants produced by exposure to various fluoroquinolones

We repeatedly exposed six Salmonella spp. strains of different serotypes (three susceptible to nalidixic acid and three resistant) to constant low concentrations of various fluoroquinolones with the aim of characterising the mutations that produce the first decrease in susceptibility to these agents. The fluoroquinolone-susceptibility of all the strains was reduced after repeated exposure to these agents. However, gyrA mutants were not always produced. Furthermore, the type of mutation produced and the time taken for it to appear varied depending on the initial resistance to nalidixic acid, the antibiotic used and the serotype involved. Therefore, we believe that the initial decrease in quinolone-susceptibility is due to various mechanisms and, in many cases, is not caused by mutations in the gyrA gene     

Trends in antimicrobial drug resistance in Salmonella enterica serotypes Typhi and Paratyphi A isolated in Europe, 1999-2001

Results of antimicrobial sensitivity tests for strains of Salmonella enterica serotypes Typhi and Paratyphi A isolated from patients in ten European countries between 1999 and 2001 have been transferred electronically to the Enter-net surveillance hub. For Typhi between 22 and 29% of isolates were multiresistant (to four drugs or more) with decreased susceptibility to ciprofloxacin (MIC 0.25-1.0 mg/l) increasing from 20% in 1999 to 26% in 2001. Nineteen of 169 (11%) strains with decreased ciprofloxacin susceptibility were sensitive to nalidixic acid. For Paratyphi A multiple resistance increased from 9% in 1999 to 25% in 2001 and decreased ciprofloxacin susceptibility from 6 to 17%. Clinicians should be aware of the possibility of treatment failures when fluoroquinolones are used as the first-line drug for infections with Typhi and Paratyphi A, particularly for patients recently returning from areas where drug-resistant strains are endemic.     

Isolation of potential bacterial pathogens from the stool of HIV-infected and HIV-non-infected patients and their antmicrobial susceptibility patterns in Jimma Hospital,south west Ethiopia

Diarrhea is a major clinical problem in HIV-infected patients. There is a need to monitor antimicrobial susceptibility patterns of enteric bacterial pathogens in order to ensure appropriate treatment and control of infections. The objectives of this study was to identify and determine the magnitude of potential enteric pathogens including Salmonella, Shigella, Campylobacter and other species in HIV-infected and HIV-non-infected patients with diarrhea, to evaluate the current antimicrobial susceptibility pattern of the clinical isolates and the association of enteric bacterial pathogens in HIV infected patients with diarrhea. A cross-sectional study was conducted from Feb-July 2001 on 372 consecutive HIV seropositive and seronegative patients presenting at Jimma hospital for different illnesses. Patients were selected based on their serological tests for HIV. Sample of faeces specimens were collected and inoculated onto standard culture media as well as onto Skirrow's medium for isolation of Campylobacter species. Salmonella and Shigella species were tested for antimicrobial susceptibility using disc agar diffusion technique recommended by Kirby-Bauer. Stool specimens were also smeared and stained by Zehl-Neelson staining technique for the identification of Mycobacterium species. Among the 99 HIV-infected patients with diarrhea, 25 (25.0%) of them had enteric bacteria among which 8(8.1%) were Salmonella, 4(4.0%) Shigella and 13(13.1%) Campylobacter species. Mycobacterium species were identified in 3(3.0%) of stool specimens obtained from HIV-infected patients with diarrhea and another 3 species were detected in HIV-infected patient without diarrhea. Salmonella species were isolated with higher prevalence in HIV-infected than in HIV non-infected patients. These Salmonella isolates were 100% susceptible to Amikacin, Gentamicin, Nalidixic acid and Kanamycin while Shigella isolates were 100% susceptible for Gentamycin and Kanamycin only. Unlike Salmonella, Shigella and Campylobacter species showed higher prevalence rates in HIV non-infected patients. Enteric bacterial pathogens account for about one fourth diarrhea in HIV infected patients in Jimma hospital. The finding of this investigation also confirmed earlier observations of wide spread resistance to the commonly used drugs in this region.     

Antibiotic resistance in Salmonella enterica subsp. enterica strains isolated in Poland from 1998 to 1999.

A total of 326 Salmonella enterica subsp. enterica strains representing 29 serotypes, isolated from human stool specimens during 1998-1999 in sanitary-epidemiological units in Poland were tested for antibiotic susceptibility by a standard disk diffusion method. The antibiotics used were ampicillin, cefotaxime, chloramphenicol, tetracycline, streptomycin, gentamicin, kanamycin, nalidixic acid, ciprofloxacin, furazolidone, cotrimoxazole, sulphonamides and trimethoprim. In addition, 201 strains belonging to the five most commonly isolated serotypes (S. Enteritidis, S. Typhimurium, S. Hadar, S. Infantis and S. Virchow) also had minimal inhibitory concentrations (MICs) determined for amoxycillin/clavulanic acid. Selected strains were screened for production of extended spectrum beta-lactamases (ESBLs). There were 49.4% of Salmonella enterica subsp. enterica strains resistant to two or more antibiotics, with the highest prevalence of multiple resistant strains among serotypes Typhimurium, Hadar and Virchow. Resistance to ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone and sulphonamides occurred most frequently. Over 93% of S. Virchow strains were resistant to furazolidone. No strains resistant to ciprofloxacin by disk-diffusion method were detected but 31.3% of isolates of the 201 strains representing the five most common serotypes had reduced ciprofloxacin susceptibility (MICs ranging 0.125-0.5 mg/l). One strain (S. Mbandaka) was resistant to cefotaxime and produced ESBL.     

Salmonella bloodstream infections: report from the SENTRY Antimicrobial Surveillance Program (1997-2001)

Salmonella spp. are significant bloodstream pathogens and are routinely monitored for antimicrobial resistance by the SENTRY Antimicrobial Surveillance Program. Six hundred and one bloodstream infection (BSI) isolates of Salmonella spp., collected over a 5-year period (1997-2001) were tested for their susceptibility against 20 antimicrobial agents, comparing year and geographical region. Salmonella enterica serotype Typhi was the most frequently identified 'species' (43% of identified strains), although 'unspeciated' strains predominated overall (54.2%). The rank order for six selected drugs tested by their MIC(90) values and percentage susceptibility was: ceftriaxone (< or =0.25 mg/l; 99.5% susceptible)>ciprofloxacin (0.12 mg/l; 99.3%)> trimethoprim/sulphamethoxazole (< or =0.5 mg/l; 92.7%)>amoxycillin/clavulanate (16 mg/l; 89.7%)>ampicillin (>16 mg/l; 81.0%)>tetracycline (>8 mg/l; 79.4%). Most isolates remained highly susceptible to all 20 agents examined, with the exception of Salmonella Typhimurium (only 35.3% susceptible to tetracycline, 41.2% to ampicillin, and 61.8% to amoxycillin/clavulanate). DT104 resistance phenotypes were noted in 3.4 and nearly 60.0% of unspeciated Salmonella and S. Typhimurium, respectively. Unexpectedly, the highest overall susceptibility rates were recorded in Latin America. Fluoroquinolone resistance was observed and nalidixic acid screening MICs (< or =8 mg/l) predicted full susceptibility to ciprofloxacin. Five-year results from the SENTRY Program show no clear trend toward greater resistances in Salmonella spp. BSIs for the commonly used antimicrobial classes. With the exception of S. Typhimurium DT104, most Salmonella spp. remain highly susceptible to the tested antimicrobials that maybe utilized for Salmonella BSI.     

Comparative in vitro activity and the inoculum effect of ertapenem against Enterobacteriaceae resistant to extended-spectrum cephalosporins

The in vitro activity and the inoculum effect of ertapenem were evaluated against a total of 70 Enterobacteriaceae isolates resistant to extended-spectrum cephalosporins. The extended-spectrum beta-lactamase phenotypic confirmatory disk diffusion test was performed and AmpC-inducible species were detected using cefoxitin/cefotaxime disk antagonism tests. beta-Lactamases were characterised by isoelectric focusing and TEM-specific polymerase chain reaction. Minimum inhibitory concentrations (MICs) were determined by the National Committee for Clinical Laboratory Standards agar dilution method. Ertapenem showed excellent activity against almost all isolates tested, with MIC(50) and MIC(90) values of 0.03 mg/L and 0.12 mg/L, respectively. When the inoculum was increased 100-fold, susceptibility decreased from 98.6% to 65.7% for cefepime and from 75.7% to 54.3% for piperacillin/tazobactam, without changing for ertapenem. The data from this study suggest that this new carbapenem may be useful for treating mixed infections involving Enterobacteriaceae isolates resistant to third-generation cephalosporins.     

临床分离志贺菌耐药性分析及1类整合子的检测

目的了解2007年至2010年安徽省临床分离志贺菌耐药性和1类整合酶基因(intⅠ1)、qacE△1-sul1基因及整合子携带的耐药基因盒的分布。方法琼脂稀释法检测21种抗菌药物对志贺菌的最低抑菌浓度。PCR扩增intⅠ1和qacE△1-sul1基因,对阳性菌株可变区基因盒序列进行分析。结果 137株志贺菌对萘啶酸、氨苄西林、磺胺甲噁唑/甲氧苄啶的耐药率均80.0%以上,且多重耐药率达94.2%,但对三代头孢菌素和氟喹诺酮类耐药率在33.0%以下。intⅠ1的检出率为89.1%(122/137),检出dfrA17-aadA5和aar-3-aacA4两种基因盒,首次在志贺菌中检测到aar-3-aacA4基因,GenBank登录号JF271916。结论安徽省临床分离志贺菌多重耐药现象严重。临床仍可选用氟喹诺酮类和三代头孢菌素作为本地区细菌性痢疾的经验性治疗。1类整合子与志贺菌的耐药具有一定的相关性。     

Colistin susceptibility testing by Etest and disk diffusion methods

The accuracy of disk susceptibility methods for colistin against 778 bacterial pathogens was evaluated in comparison with Etest using interpretive criteria available from the Clinical and Laboratory Standards Institute (CLSI). Colistin exhibited excellent activity against Acinetobacter baumannii and Escherichia coli isolates (minimum inhibitory concentration for 90% of the organisms (MIC₉₀) = 0.5 mg/L), whilst it was less active both against Enterobacter spp. and Klebsiella pneumoniae (MIC for 50% of the organisms (MIC₅₀) = 0.5 mg/L, MIC₉₀ = 16 mg/L). Colistin also showed good activity against Pseudomonas aeruginosa (MIC₉₀ = 2 mg/L, MIC₅₀ = 1 mg/L) but poor activity against Stenotrophomonas maltophilia (MIC₅₀ = 8 mg/L, MIC₉₀ = 128 mg/L). Only 0.8% of minor errors were observed between the studied methods for P. aeruginosa isolates when the CLSI criteria were applied. All A. baumannii isolates with a zone diameter ≤12 mm were resistant and those with a zone diameter ≥14 mm were susceptible according to MIC breakpoints established by the CLSI. Among nine isolates exhibiting a zone diameter of 13 mm, one was resistant to colistin (MIC = 8 mg/L) and eight isolates were susceptible (MIC = 0.5 mg/L). Applying a MIC breakpoint of ≤2 mg/L for susceptibility in Enterobacteriaceae, all isolates with a zone diameter ≥14 mm were susceptible, whilst all isolates with a zone diameter ≤11 mm were resistant. Among isolates with zone diameters of 12–13 mm, 59% were characterised as susceptible. Major errors were observed only in K. pneumoniae isolates at a rate of 0.8%. The poor agar diffusion characteristics of colistin limit the predictive accuracy of the disk diffusion test and consequently values of 12–13 mm should be confirmed with MIC determination by Etest or broth dilution method.     

Available options in the management of non-typhi Salmonella

Gastroenteritis, caused by Salmonella spp. is usually a self-limiting infection and does not require treatment. However, in some immunosuppressed patients (such as the newborn, the elderly, those with AIDS or neoplasms), there is a greater risk of developing a severe systemic infection, and in these cases, antibiotic treatment is recommended. Third-generation cephalosporins and fluoroquinolones are the most useful antibiotics in the treatment of these infections, although resistant strains are sometimes isolated. Therapeutic failures have been reported with fluoroquinolones in extra-intestinal infections caused by nalidixic acid resistant strains.     

Available options in the management of non-typhi Salmonella

Gastroenteritis, caused by Salmonella spp. is usually a self-limiting infection and does not require treatment. However, in some immunosuppressed patients (such as the newborn, the elderly, those with AIDS or neoplasms), there is a greater risk of developing a severe systemic infection, and in these cases, antibiotic treatment is recommended. Third-generation cephalosporins and fluoroquinolones are the most useful antibiotics in the treatment of these infections, although resistant strains are sometimes isolated. Therapeutic failures have been reported with fluoroquinolones in extra-intestinal infections caused by nalidixic acid resistant strains.