Accuracy of the disk method for determining antimicrobic susceptibility of common Gram-negative bacilli

A standardized disk susceptibility test was evaluated by comparing results with minimal inhibitory concentrations obtained with agar dilution methods. The agar overlay method was used to test 152 Gram-negative bacilli against eight different antimicrobial agents. One to 3% of the isolates were resistant to an antimicrobic by the MIC method, but appeared to be susceptible by the disk method. Most very major discrepancies involved disk tests withProteus sp., a microorganism notoriously difficult to test reproducibly.Serratia sp. vs. the polymyxins andKlebsiella sp. vs. nitrofurantoin accounted for most other major discrepancies. With other microorganism-drug combinations, the disk test was a reasonably accurate technique for classifying bacteria into resistant or susceptible categories. Gentamicin disk tests were unsatisfactory, but when an intermediate zone category of 13–16 mm was applied, the false susceptible test results were reduced to 2.6%. Intermediate zone sizes were obtained with 6% of the disk tests; most of those isolates were resistant or susceptible but not intermediate in susceptibility. About 11% of the strains had intermediate MICs (5–20% with different drugs), but most of those strains were fully susceptible by the disk technique. *** DIRECT SUPPORT *** A01R4011 00007     

Antimicrobial susceptibility and molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> from cholera outbreaks in Chennai

The genotype and antibiotic resistance pattern of the toxigenic Vibrio cholerae strains associated with cholera outbreaks vary frequently. Fifty-one V. cholerae strains isolated from cholera outbreaks in Chennai (2002–2005) were screened for the presence of virulence and regulatory genes by multiplex polymerase chain reaction (PCR) assay. Genotyping of the isolates was done by VC1 primers derived from enterobacterial repetitive intergenic consensus (ERIC)-related sequence in V. cholerae. All the isolates possessed toxigenic genes, such as ctxA, ctxB, tcpA, ace, ompU, toxR and zot. Two different El Tor genotypes and one O139 genotype could be delineated by VC1-PCR. One of the El Tor genotypes was similar to the El Tor strains isolated from Bhind district and Delhi during 2004. Antibiotic susceptibility testing revealed greater variability among the isolates tested. All the isolates were found to be susceptible to norfloxacin, ciprofloxacin and tetracycline. Thiry-three per cent of the isolates were found to be resistant to more than 4 antibiotics and could be termed as multiple antibiotic resistant. Coexistence of O139 serogroup along with the El Tor biotype could be identified among the strains recovered during the period 2002–2004. The O139 isolates were found to be more susceptible to the antibiotics tested when compared to the El Tor isolates.     

Synergistic activity of 1-(1-naphthylmethyl)-piperazine with ciprofloxacin against clinically resistant Staphylococcus aureus, as determined by different methods

Aims: To evaluate the interaction of 1‐(1‐naphthylmethyl)‐piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)‐resistant clinical isolates of methicillin‐resistant Staphylococcus aureus (MRSA). Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQ‐resistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ‐resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time–killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first‐line antimicrobial agent CPFX against Staph. aureus. Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ‐resistant Staph. aureus was observed in vitro. Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX.     

Antimicrobial resistance and species identification of staphylococci isolated from the meat of wild rabbits (<Emphasis Type="Italic">Oryctolagus cuniculus</Emphasis>) in Slovakia

In 2009, a total of 113 strains of staphylococci were isolated from the thigh muscles of ten hunted and 20 farmed wild rabbits (Oryctolagus cuniculus) in the Slovak Republic. Only two isolates (1.8%) possessed coagulase activity, the rest of 111 staphylococcal isolates were coagulase-negative. Among them, six isolates (5.4%) showed the production of DNase. In each isolate, resistance to eight antibiotics by means of agar dilution test was tested. Based on these results, 110 isolates were found to be resistant to at least one antibiotic. Only one isolate was susceptible to all eight antibiotics tested. Another two isolates were susceptible, however, they showed intermediate susceptibility to cefoxitin. Resistance to ampicillin (78.8%), erythromycin (58.4%), penicillin (51.3%) and oxacillin (46.0%) was found most frequently. Twenty-six isolates (23.0%) were resistant to novobiocin. On the other hand, resistance to cefoxitin (8.0%) and gentamicin (1.8%) were quite rare. Fifteen percent of isolates were resistant to one antibiotic, simultaneous resistance to two, three, four and five antibiotics was confirmed in 22.1%, 23.9%, 21.2% and 13.3% of isolates, respectively. Except for two coagulase-positive Staphylococcus aureus isolates (1.8%), seven species of coagulase-negative staphylococci were identified using the MALDI BioTyper (TM) sytem as follows: Staphylococcus warneri (45.1%), Staphylococcus epidermidis (21.2%), Staphylococcus pasteuri (13.3%), Staphylococcus xylosus (8.0%), Staphylococcus capitis (7.1%), Staphylococcus haemolyticus (1.8%) and Staphylococcus cohnii ssp cohnii (1.8%).     

<Emphasis Type="Italic">In Vitro</Emphasis> activity of telithromycin and quinupristin/dalfopristin against methicillin-resistant coagulase-negative staphylococci with defined resistance genotypes

We determined the activities of new antibiotics telithromycin (ketolide) and quinupristin/dalfopristin (streptogramins) against 88 macrolide and/or lincosamide resistant coagulase-negative staphylococci (CoNS) isolates with defined resistance gene status. Telithromycin susceptibility was determined only in erythromycin-sensitive isolates (15) indicating the same mechanisms of resistance. In contrast, all erythromycin-resistant isolates (73) were either constitutively resistant to telithromycin (13 isolates with constitutive erm genes) or demonstrated telithromycin D-shaped zone (60 isolates with inducible msr(A) and/or erm). However, the level of inducible resistance conferred by msr(A) (35 isolates) was borderline even after induction by erythromycin. No quinupristin/dalfopristin resistant isolate was observed if tested by disk-diffusion method (DDM) but 18 isolates were intermediate (MIC = 1-3 mg/L) and two isolates resistant (MIC = 8 mg/L) if tested by E-test. All these isolates were resistant to streptogramin A and harbored vga(A) gene (1 isolate) or vga(A)LC gene (19 isolates). MICs for quinupristin/dalfopristin were higher for isolates with combination of streptogramin A resistance and constitutive MLSB resistance (MIC = 3-8 mg/L in 4 isolates) than for streptogramin A-resistant isolates susceptible to streptogramin B (MIC = 0.5-2 mg/L in 16 isolates). In addition to S. haemolyticus, vga(A)LC was newly identified in S. epidermidis and S. warnerii indicating its widespread occurrence in CoNS. Misidentification of low-level resistant isolates by DDM may contribute to dissemination of streptogramin A resistance.     

肺炎克雷伯菌gyrA基因与parC基因的突变研究

目的探讨肺炎克雷伯菌对环丙沙星和左氧氟沙星的药物敏感性,及对喹诺酮敏感和耐药菌株中gyrA与parC基因的突变情况。方法收集肺炎克雷伯菌临床分离株231株,采用K-B纸片法测定肺炎克雷伯菌对环丙沙星和左氧氟沙星的敏感性,随机选取对环丙沙星和左氧氟沙星均耐药菌株4株和均敏感的菌株3株,分别PCR扩增gyrA基因和parC基因的耐药决定区,扩增片段长度分别为625、319bp,PCR扩增产物经纯化后测序并做序列分析。结果肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药率分别为51.1%（118/231）和45.9%（106/231）;gyrA和parC基因经序列分析显示,耐药株均有gyrA基因的突变,其中1株出现第83、87和27位氨基酸的改变,2株出现第83位氨基酸的改变,1株出现第47位点的改变;环丙沙星敏感株中未出现gyrA基因的突变。4株耐药株均有parC基因的突变,引起相应氨基酸Ser80→Arg的改变,2株环丙沙星敏感株也发生了同样的改变。结论哈尔滨地区肺炎克雷伯菌对环丙沙星和左氧氟沙星的耐药性显著,在喹诺酮耐药株中有gyrA和parC基因的同时突变,在敏感株中也发现了parC基因的突变。     

Expression of efflux pump gene lde in ciprofloxacin‐resistant foodborne isolates of Listeria monocytogenes

The expression of efflux pump gene lde in ciprofloxacin resistant (Cip^R) and susceptible strains of Listeria monocytogenes collected from retail food samples was investigated. For two Cip^R strains, the MICs of ciprofloxacin decreased four‐ to eightfold in the presence of reserpine; however, no significant alterations were observed with naturally sensitive isolates. Overexpression of the lde gene induced by ciprofloxacin was observed in two resistant isolates. The present findings indicate that expression of lde and the MICs of ciprofloxacin are well correlated with the presence and absence of reserpine, suggesting that Lde might be involved in ciprofloxacin resistance of L. monocytogenes.     

Multicenter evaluation of Neo-Sensitabs, a standardized diffusion method for yeast susceptibility testing

The agar diffusion method Neo-Sensitabs for sensitivity testing, was evaluated with 33 reference strains by fourteen laboratories. Tablets with 5-fluorocytosine, amphotericin B, nystatin, fluconazole, itraconazole, ketoconazole and tioconazole were used on Shadomy modified medium. These tests classify each strain as susceptible, intermediate or resistant to all tested antifungals by measuring the inhibition zone diameters. Intra and interlaboratory reproducibility was studied. Neo-Sensitabs sensitivity for fungi was easy to perform and reliable method with a reproducibility of 97.1% and superior to other commercialized methods, being specially interesting for antifungal susceptibility in vitro testing of triazole derivatives fluconazole and itraconazole.     

Susceptibility of Arcobacter butzleri isolates to 23 antimicrobial agents

AIMS: The objective of this study was to determine the susceptibility of Arcobacter butzleri isolates to various antimicrobial agents used in the treatment of infectious diseases in humans and animals. METHODS AND RESULTS: Thirty-nine A. butzleri strains isolated from broiler chickens were tested for their susceptibility to 23 antimicrobial agents using a disc diffusion method. All isolates were resistant to aztreonam, cefuroxime sodium, cephalothin, orbenin, oxacillin, penicillin G and trimethoprim/sulphamethoxazol. Of the 39 isolates tested, 26 were also found resistant to amoxycillin, amoxycillin/clavulanic acid and ampicillin. One isolate was resistant to, and four showed intermediate level of resistance to, erythromycin. All isolates were susceptible to amikacin, chloramphenicol, danofloxacin, enrofloxacin, nitrofurantoin, nalidixic acid, tetracyclines and tobramycin. CONCLUSIONS: The majority of the isolates were found resistant to antibiotics commonly used for the treatment of infectious bacterial diseases in humans and animals. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that A. butzleri strains vary in their resistance to certain kinds of antibiotics and caution should be taken when choosing a suitable antibiotic for the treatment of disease(s) caused by this organism.     

Effects of Antifungal Agents in Sap Activity of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates

Some antifungal agents have shown to exert effects on expression of virulent factors of Candida as the production of secretory aspartyl proteinase (Sap). In this study, we sought to determine and to compare the influence of fluconazole and voriconazole in proteinase activity of this microorganism. Thirty-one isolates obtained from oral mucosa of human immunodeficiency virus positive (HIV⁺) patients were used in this study. The minimal inhibitory concentrations (MIC) of fluconazole and voriconazole were determined using the broth microdilution method with RPMI 1640 medium and with yeast carbon base–bovine serum albumin (YCB–BSA) medium. The Sap activity following by digestion of BSA as substrate was determined for four Candida albicans strains arbitrarily chosen according to susceptibility (susceptible or resistant) to fluconazole or voriconazole. Besides, the SAP1 to SAP7 genes were screened by PCR for the same isolates that were determined by the Sap activity. In vitro susceptibility testing using the two media presented similar MIC values. Increased Sap activity was observed in resistant isolates on presence of drugs, but the Sap activity by susceptible isolates to azoles showed different behavior on the presence of drug. We detected the presence of SAP1 to SAP7 genes from all susceptible or resistant C. albicans isolates. The present study provides important data about the proteinase activity and the presence of genes of SAP family in fluconazole and voriconazole susceptible or resistant C. albicans isolates.     

Infection of cereals and grasses by isolates of Polymyxa graminis (Plasmodiophorales)

The host range of isolates of Polymyxa was tested in mono-fungal sand cultures. Fourteen isolates of P. graminis, obtained from barley, wheat, oats or Poa annua and from several different countries, all infected barley and all but one infected wheat. Rye was also a good host, whereas oats (nine cultivars), Lolium multiflorum and Poa pratensis became only slightly infected. Wheat cultivars differed in susceptibility, with Galahad much more resistant than Avalon. Several common weed and pasture grasses were not infected by the two isolates tested. A range of wild Hordeum spp. were mostly susceptible to P. graminis and/or barley mild mosaic virus, which it transmits. An isolate of P. betae, used for comparison, caused slight infection on oats but not on other cereals. The variation within and between species of Polymyxa needs more detailed investigation.     

Genetic mapping of the novel <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> resistance gene <Emphasis Type="Italic">TuRB03</Emphasis> in <Emphasis Type="Italic">Brassica napus</Emphasis>

A new source of resistance to the pathotype 4 isolate of Turnip mosaic virus (TuMV) CDN 1 has been identified in Brassica napus (oilseed rape). Analysis of segregation of resistance to TuMV isolate CDN 1 in a backcross generation following a cross between a resistant and a susceptible B. napus line showed that the resistance was dominant and monogenic. Molecular markers linked to this dominant resistance were identified using amplified fragment length polymorphism (AFLP) and microsatellite bulk segregant analysis. Bulks consisted of individuals from a BC₁ population with the resistant or the susceptible phenotype following challenge with CDN 1. One AFLP and six microsatellite markers were associated with the resistance locus, named TuRB03, and these mapped to the same region on chromosome N6 as a previously mapped TuMV resistance gene TuRB01. Further testing of TuRB03 with other TuMV isolates showed that it was not effective against all pathotype 4 isolates. It was effective against some, but not all pathotype 3 isolates tested. It provided further resolution of TuMV pathotypes by sub-dividing pathotypes 3 and 4. TuRB03 also provides a new source of resistance for combining with other resistances in our attempts to generate durable resistance to this virus.     

Determination of the Efflux Pump-Mediated Resistance Prevalence in Pseudomonas aeruginosa, Using an Efflux Pump Inhibitor

In Gram negative bacteria, fluoroquinolone resistance is acquired by target mutations in topoisomerase genes or by reducing the permeation of drugs due to the increase in expression of endogenous multidrug efflux pumps that expel structurally unrelated antimicrobial agents. An ongoing challenge is searching for new inhibitory substances in order to block efflux pumps and restore the antibiotic drugs susceptibility. In this research, we sought to investigate the interplay between ciprofloxacin and an efflux pump inhibitor (EPI), phenyl alanine arginyl β-naphtylamide (PAβN), to determine the prevalence of efflux pump overexpression in clinical isolates of Pseudomonas aeruginosa. Ciprofloxacin was tested at different concentrations (256–0.25 μg/ml) with a fixed concentration of PAβN (50 μg/ml). The isolates susceptibility profiles were analyzed by disc diffusion and agar dilution methods using 10 antibiotic discs and 4 powders. It was found that in the presence of PAβN, resistance to ciprofloxacin was inhibited obviously and MIC values were decreased. The comparison between subgroups of P. aeruginosa isolates with different resistance profiles indicates that efflux pump overexpression (EPO) is present in 35% of ciprofloxacin resistant isolates with no cross resistance and in variable frequencies among isolates showing cross resistance to other tested antibiotics: gentamicin (31%), ceftazidime (29%), and imipenem (18%). Altogether, these results imply that PAβN maybe effective to restore the fluoroquinolone drugs susceptibility in clinical treatment procedures. Results also show that increased use of a fluoroquinolone drug such as ciprofloxacin can affect the susceptibility of P. aeruginosa to other different antipseudomonal agents.     

The comparative activity of cefepime and other current antibiotics against microorganisms isolated from patients in pediatric intensive therapy units]

Comparative activity of cefepime, ceftazidime, ceftriaxone, ciprofloxacin, imipenem and piperacillin/tazobactam against isolates from patients of pediatric intensive care units within October-December 1998 was studied. The isolates were identified with the Walkaway-40 System. The antibiotic susceptibility was evaluated by the E-test on the Mueller-Hinton II agar. The data were interpreted in accordance with the NCCLS. The number of the isolates totaled 100. Among them not more than 10 to 12 strains belonged to the same species. 92 per cent of the isolates was susceptible to cefepime and 70 to 75 per cent of the isolates were susceptible to the third generation cephalosporins. Piperacillin/tazobactam proved to be highly susceptible, the number of the isolates resistant and moderately resistant to it being 6 and 1 per cent respectively. The results showed that cefepime was intermediate between the third generation cephalosporins and carbapenems.     

Monitoring of resistance genes in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> isolates and their presence in the extracellular DNA of biofilms: a case study from the Czech Republic

The alarming occurrence of antibiotic resistance genes in food production demands continuous monitoring worldwide. One reservoir of resistance genes is thought to be eDNA. There is currently little available information in Europe about either the extracellular DNA distribution of the bacterium or the spread of resistance genes in L. monocytogenes. Therefore, our aims were to give insight into the Listeria monocytogenes resistance situation in the Czech Republic and assess the presence of resistance genes in their extracellular DNA (eDNA). First, susceptibility tests were performed on 49 isolates of L. monocytogenes with selected antibiotics. Next, we tested DNA of suspected isolates for the presence of resistance genes in both planktonic cells and the eDNA of biofilms. Finally, fluorescent confocal microscopy was used to observe the eDNA pattern of selected isolates under conditions that mimicked the food processing environment and the human body. Susceptibility tests found isolates intermediate resistant to chloramphenicol, tetracycline, and ciprofloxacin as well as isolates resistant to ciprofloxacin. For all suspected isolates, PCR confirmed the presence of the gene lde encoding efflux pump in both types of DNA. When the biofilm was observed using confocal laser scanning microscope, the eDNA distribution patterns varied considerably according to the culture conditions. Furthermore, the food and clinical isolates varied in terms of the amount of eDNA detected. The presence of an efflux pump in both types of DNA suggests that the eDNA might serve as a reservoir of resistance genes. Surprising differences were observed in the eDNA pattern. Our results suggest that the current risk of the spread of L. monocytogenes resistance genes is low in the Czech Republic, but they also indicate the need for continuous long-term monitoring of the situation.     

Comparison of the accuracy of disk diffusion zone diameters obtained by manual zone measurements to that by automated zone measurements to determine antimicrobial susceptibility

Although a variety of techniques are available for antimicrobial susceptibility testing, disk diffusion methods remain the most widely used. We compared the accuracy of disk diffusion zone diameters as obtained by manual zone measurements in a low resource country (Indonesia) to that by automated zone measurements (Oxoid aura image system) in a high resource setting (the Netherlands) to determine susceptibility categories (sensitive, intermediate susceptible or resistant). A total of 683 isolates were studied, including 294 Staphylococcus aureus, 195 Escherichia coli and 194 other Enterobacteriaceae. Antimicrobial agents included tetracycline, oxacillin, gentamicin, erythromycin, trimethoprim/sulfamethoxazole and chloramphenicol for S. aureus and ampicillin, gentamicin, cefotaxime, ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol for E. coli and other Enterobacteriaceae. Of the 4098 drug-organism combinations, overall category agreement (CA), major discrepancy (MD) and minor discrepancy (mD) between the two methods were 82.4% (3379/4098), 6.0% (244/4098) and 11.6% (475/4098), respectively. One hundred and sixty three of 244 MDs were resolved using reference broth microdilution method. Overall very major error (VME), major error (ME) and minor error (mE) of manual zone measurement were 28.8%, 45.4% and 4.9%, respectively and for the aura image system 4.9%, 16.0% and 4.9%, respectively. The results of this study indicate that the disk diffusion method with manual zone measurement in Indonesia is reliable for susceptibility testing. The use of an automated zone reader, such as the aura image system, will reduce the number of errors, and thus improve the accuracy of susceptibility test results for medically relevant bacteria.     

Multiresistance of Staphylococcus xylosus and Staphylococcus equorum from Slovak Bryndza cheese

Staphylococcus xylosus, Staphylococcus equorum, and Staphylococcus epidermidis strains were isolated from Bryndza cheese and identified using PCR method. The antimicrobial susceptibility of these strains was assessed using disc diffusion method and broth microdilution method. The highest percentage of resistance was detected for ampicillin and oxacillin, and in contrary, isolates were susceptible or intermediate resistant to ciprofloxacin and chloramphenicol. Fourteen of the S. xylosus isolates (45 %) and eleven of the S. equorum isolates (41 %) exhibited multidrug resistance. None of the S. epidermidis isolate was multiresistant. The phenotypic resistance to oxacillin was verified by PCR amplification of the gene mecA.     

Susceptibility to chemotherapeutics and the occurrence of metallo-beta-lactamases in hospital Pseudomonas aeruginosa strains

The aim of the study was evaluation of susceptibility of Pseudomonas aeruginosa strains isolated from patients hospitalized in different wards of Rydygier's Hospital in Krakow in 2005. Bacteria were identified on the basis of typical morphology confirmed by Gram-staining microscopy and by biochemical tests--ID 32 GN strips using ATB system (bioMerieux, France). The susceptibility of all isolates to a panel of antimicrobial agents was performed using disk diffusion method. The highest in vitro activity against clinical strains demonstrated ceftazidime (88.6% of susceptible strains) while the lowest in vitro activity against clinical strains demonstrated imipenem (50.4% of susceptible strains). It was also observed that 40.2%(53) of strains were resistant to meropenem and imipenem. Carbapenem resistant P aeruginosa strains were tested for MBL production. We performed disk synergy test for MBL detection with EDTA, 2-MPA and ceftazidime, imipenem. The presence of a distorted inhibition zone was interpreted as a positive result for MBL production. Positive results of disk synergy tests were confirmed by Etest MBL strips. Metalo-beta-lactamases were detected in 13 isolates resistant to carbapenems.     

Antibiotic resistant N. gonorrhoeae in Trinidad and Tobago.

We tested the susceptibility patterns of 128 N. gonorrhoeae isolates to six antimicrobials; penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin and azithromycin, and examined whether certain demographic or behavioral factors related to antibiotic use increased the likelihood of infection by a resistant strain. There was a low rate of resistance to penicillin; penicillinase-producing and chromosomal-mediated penicillin resistant gonorrhea were estimated to be 0.8%. A much higher proportion of isolates were resistant to tetracycline (up to 15%). All isolates were sensitive to spectinomycin, ciprofloxacin and ceftriaxone. However, less than 2% of isolates displayed intermediate resistance to both ciprofloxacin and ceftriaxone, and 9% exhibited intermediate resistance to spectinomycin. Patients who had obtained medication before attending the clinic and had taken all of the medication were more likely (p = 0.03) to be infected with a resistant strain of gonococcus. Also, patients who were asked by a clinic doctor to return for a test of cure during an earlier clinic visit, but who did not return were more likely to be infected with a resistant organism (p = 0.006) compared to those who returned at the doctor's request. These findings have important implications for antibiotic use and educational programs in Trinidad and Tobago.