Development and Transduction of Rsistance to Novobiocin and Nikkol-SNP 7.5 A (an Anionic Surfactant) in Staphylococcus aureus

The susceptibility to Novobiocin (NB) and Nikkol-SNP 7.5 A (NS), an anionic surfactant, was studied in 458 strains of coagulase-positive Staphylococcus aureus isolated from clinical sources. Twenty three (4.9%) of these strains were resistant to NB with minimum inhibition concentration (MIC) of 1.6 μg per ml or more, 97 (21.2%) resistant to NS with MIC of 6.25 mg per ml or more, and 17 (3.7%) resistant to both drugs. Cross-resistance to NB and NS was found in 74 per cent of 23 NB-resistant strains and 17.5 per cent of 97 NS-resistant strains. Nearly one half of NS-resistant strains belonged to phase group I, while the remainder were non-typable. The majority of the NB-resistant strains were not phage typable. In S. aureus strain PS 53 used for propagating phage 53, resistance to 25 mg per ml of NS was attained rapidly by single step without accompanying that to NB, whereas resistance to 25 μg per ml of NB developed gradually by three successive steps and was accompanied by a rapid development of resistance to NS first by two steps. The transduction experiments in strain PS 53 showed that resistance to NB and NS was jointly transduced and the genetic loci responsible for resistance to both drugs are closely linked.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

Discrepancies between MIC and MLC values of amphotericin B against isolates ofAspergillus species

There is little information addressing the phenomena of discrepancy between minimal inhibitory concentrations (MIC) and minimal lethal concentrations (MLC) values of amphotericin B (AMB) to clinical isolates of fungi. This study assessed in vitro activity of AMB against 70 clinical isolates of aspergilli: 30 strains ofAspergillus fumigatus, 20 strains ofAspergillus flavus and 20 strains ofAspergillus niger. Susceptibility tests were accomplished using a macro broth dilution procedure, with special emphasis on ascertainment of MLCs. AMB exhibited low MIC values against all clinical isolates. While we did not identify any AMB resistant isolates among 70Aspergillus spp. studied as judged by MIC levels, analysis of the data demonstrated a clear discrepancy between the MIC and MLC levels of AMB obtained against clinical isolates ofAspergillus spp. The MLC values of AMB were significantly higher than the MIC values with MIC 50 and MIC 90 of 0.29 and 0.5 µg/ml, respectively, at the second reading time, and MLC 50 and MLC 90 of 2.31 and 9.24 µg/ml, respectively (p<0.001). Additionally, minimal lethal concentrations in 36/70 (51%) of aspergillal isolates studied produced drug concentrations above those which can usually be sustained in patient plasma or tissue.     

Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.     

Mycotoxin producing potential ofFusarium graminearum isolates from Uruguayan barley

Summary The activity of antimicrobial agents against clinical isolates ofNocardia was studied by determination of the Minimal Inhibitory Concentrations (MICs) and Disk Diffusion Technique, according to the National Committee for the clinical Laboratory Standards (NCCLS). The object was: (a) to determine the in vitro susceptibility of the strains that cause human mycetomas; (b) to determine the presence of different patterns of sensitivity among the regional strains; (c) to evaluate the Disk Diffusion Technique using disks commercially available with the antimicrobial concentrations normally used in the microbiological practice. Comparing the MIC values obtained with the values suggested by the National Committee for Clinical Laboratory Standards forNocardia spp. (broth microdilution MIC breakpoints), we found that local strains are susceptible to amikacin, cefotaxime, ceftriaxone and TMP-SMZ; moderately susceptible to ampicillin and resistant to ciprofloxacin and erythromycin. The results obtained by both methods showed the presence of different patterns of sensitivity among the regional strains ofN. brasiliensis. This showed strains sensitive and resistant to antibiotics. The Disk Diffusion Technique, even if it is not the adequate method to study the sensitivity patterns of different strains against antimicrobial agents, permits the differentiation between strains sensitive and resistant to antibiotics.     

Association of MexAB-OprM with intrinsic resistance ofPseudomonas aeruginosa to aminoglycosides

MexAB-OprM is known to pump out mostly lipophilic and amphiphilic drugs. But in low-ionic-strength medium, nutrient broth (NB), this pump has been shown to contribute to hydrophilic antibiotic (aminoglycosides) resistance, via active efflux. The association of the MexAB-OprM efflux system to aminoglycosides resistance inPseudomonas aeruginosa were assessed using a drug susceptibility test carried out in NB, in presence and absence of protonophore, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) by 23 multidrug resistant strains were selected from 104 clinical isolates ofP. aeruginosa. Active efflux was assessed using EtBr accumulation assays. PCR was used to identify themexAB-oprM and MexAB-OprM-dependent efflux of aminoglycosides and the results were confirmed by continuous fluorescence assay. A multidrug resistant mutant ofmexAB-oprM, derivative of PAO1, was selected by ciprofloxacin and subjected to the same analysis as described above for the clinical isolates. In this study, CCCP reduced the level of MICs in at least 1 dilution. Ethidium bromide accumulation assays confirmed the presence of efflux mechanism in all clinical isolates and PCR demonstrated that 17% of our isolates had themexAB-oprM operon. Results of aminoglycosides accumulation showed, in addition to amphiphilic antibiotics in NB medium, MexAB-OprM extrudes aminoglycosides (hydrophilic) drugs.     

In silico prediction of drug resistance due to S247R mutation of Influenza H1N1 neuraminidase protein

We present here in silico studies on antiviral drug resistance due to a novel mutation of influenza A/H1N1 neuraminidase (NA) protein. Influenza A/H1N1 virus was responsible for a recent pandemic and is currently circulating among the seasonal influenza strains. M2 and NA are the two major viral proteins related to pathogenesis in humans and have been targeted for drug designing. Among them, NA is preferred because the ligand-binding site of NA is highly conserved between different strains of influenza virus. Different mutations of the NA active site residues leading to drug resistance or susceptibility of the virus were studied earlier. We report here a novel mutation (S247R) in the NA protein that was sequenced earlier from the nasopharyngeal swab from Sri Lanka and Thailand in the year 2009 and 2011, respectively. Another mutation (S247N) was already known to confer resistance to oseltamivir. We did a comparative study of these two mutations vis-a-vis the drug-sensitive wild type NA to understand the mechanism of drug resistance of S247N and to predict the probability of the novel S247R mutation to become resistant to the currently available drugs, oseltamivir and zanamivir. We performed molecular docking- and molecular dynamics-based analysis of both the mutant proteins and showed that mutation of S247R affects drug binding to the protein by positional displacement due to altered active site cavity architecture, which in turn reduces the affinity of the drug molecules to the NA active site. Our analysis shows that S247R may have high probability of being resistant.     

Tuberculate spore formation by thirty-two strains of Histoplasma capsulatum

Summary 1. Thirty-two strains ofHistoplasma capsulatum were studied concerning their ability to form tuberculate spores and their conversion into the yeast phase.2. Nine strains did not produce tuberculate spores on Sabouraud's agar, on corn meal agar, on spent medium, on media with pH adjusted from 4.5 to 7.0 or on the first passage through hamsters.3. Tuberculate spore production did occur in these nine strains when Sabouraud's medium was enriched with phosphate, especially KH₂PO₄. In addition, all but two strains produced tuberculate spores after a second passage through hamsters.4. Growth on KH₂PO₄ enriched Sabouraud's agar led to a greater yield of yeast phase as compared to yeast phase resulting from colonies of the same strain grown on plain Sabouraud's agar. This may be due to the greater number of spores produced on the KH₂PO₄ enriched medium.5. A grinding technique of preparing inocula improved slightly the facility of obtaining the yeast phase over heavy inoculation with unground pieces of mold culture.     

Subinhibitory concentrations of nalidixic acid alter bacterial physiology and induce anthropogenic resistance in a commensal strain of Escherichia coli in vitro

The human gut houses a complex group of bacterial genera, including both opportunistic pathogens and commensal micro-organisms. These are regularly exposed to antibiotics, and their subinhibitory concentrations play a pivotal role in shaping the microbial responses. This study was aimed to investigate the effects exerted by sub-MICs of nalidixic acid (NA) on the growth rate, bacterial motility, biofilm formation and expression of outer membrane proteins (OMPs) in a commensal strain of E. coli. The NA-sensitive strain was sequentially passaged under sub-MICs of NA. E-test was used to determine the MIC values of NA. Results indicated significant changes in the growth profile of commensal E. coli upon exposure to NA at sub-MICs. Differential expression of OMPs was observed in cells treated with sub-MICs of NA. Bacterial motility was reduced under 1/2 MIC of NA. Interestingly, successive passaging under 1/2 MIC of NA led to the emergence of resistant E. coli with an increased MIC value of 64 µg ml⁻¹ in just 24 days. The NA-resistant variant was confirmed by comparing its 16S rRNA sequence to that of the sensitive commensal strain. Mutations in the Quinolone Resistance-Determining Regions (QRDRs) of chromosomal gyrA, and Topoisomerase IV-encoding parC genes were detected in NA-resistant E. coli. Our results demonstrate how antibiotics play an important role as signalling molecules or elicitors in driving the pathogenicity of commensal bacteria in vitro.     

Biofilm formation by and antifungal susceptibility of Candida isolates from urine

Biofilm formation (BF) in the setting of candiduria has not been well studied. We determined BF and MIC to antifungals in Candida spp. isolates grown from urine samples of patients and performed a retrospective chart review to examine the correlation with risk factors. A total of 67 Candida spp. isolates were grown from urine samples from 55 patients. The species distribution was C. albicans (54%), C. glabrata (36%), and C. tropicalis (10%). BF varied greatly among individual Candida isolates but was stable in sequential isolates during chronic infection. BF also depended on the growth medium and especially in C. albicans was significantly enhanced in artificial urine (AU) compared to RPMI medium. In nine of the C. albicans strains BF was 4- to 10-fold higher in AU, whereas in three of the C. albicans strains and two of the C. glabrata strains higher BF was measured in RPMI medium than in AU. Determination of the MICs showed that planktonic cells of all strains were susceptible to amphotericin B (AMB) and caspofungin (CASPO) and that three of the C. glabrata strains and two of the C. albicans strains were resistant to fluconazole (FLU). In contrast, all biofilm-associated adherent cells were resistant to CASPO and FLU. The biofilms of 14 strains (28%) were sensitive to AMB (MIC(50) of <1 mug/ml). Correlation between degree of BF and MIC of AMB was not seen in RPMI grown biofilms but was present when grown in AU. A retrospective chart review demonstrated no correlation of known risk factors of candiduria with BF in AU or RPMI. We conclude that BF is a stable characteristic of Candida strains that varies greatly among clinical strains and is dependent on the growth medium. Resistance to AMB is associated with higher BF in AU, which may represent the more physiologic medium to test BF. Future studies should address whether in vitro BF can predict treatment failure in vivo.     

Determination of Drug Resistance of Mycobacterium tuberculosis Cultures

A total of 3,303 strains of Mycobacterium tuberculosis were tested for sensitivity to streptomycin (SM), isoniazid (INH), and p-aminosalicylic acid (PAS) by the Steenken modified minimal inhibitory concentration (MIC) test. A simultaneous double blind comparison was carried out on 277 selected strains by the Steenken MIC test and the Canetti proportion method. Agreement between the results for the two tests was 82% for SM, 95% for INH, and 89% for PAS. A small number of strains appeared to be sensitive when tested by one method but resistant by the other. MIC determinations were carried out on 83 strains by using Steenken-Smith, Lowenstein-Jensen, and Middlebrook 7H10 media containing a more extended range of concentrations of the test drugs. The MIC values for both SM and dihydrostreptomycin increased on Steenken-Smith medium compared with the other two. INH did not show any medium effect, whereas PAS showed increased MIC values in 7H10 agar. The significance of the comparisons of the MIC values on the various media is discussed in terms of possible changes in the drug sensitivity testing methods used at present in this laboratory.     

Plasmid-linked drug resistance in Salmonella typhimurium in Kuwait

A total of 143 strains of S. typhimurium isolated from clinical cases in Kuwait were investigated for drug resistance. Multidrug resistance (3 or more drugs) was seen in 76.9% of the strains. The common resistance patterns were ASuT (16), AKSuT (20), ASSuT (14), CKSSuT (14), ACKSSuT (10), ACGKSSuT (15) and ACKGSSuTTm (8). MIC of resistant strains was usually high. Sixteen isolates were examined for the transferability of the resistance. All had R-plasmids, both autotransferable and non-autotransferable which could be mobilised by factor X.     

The kinetics of photoinactivation and long-term photosensitivity of pigmented and nonpigmented strains of the smut fungusUstilago violacea

White, pink, pumpkin, and yellow strains ofUstilago violacea containing high and low levels of cytochrome c and various carotenes were subjected to high light intensities to characterize the kinetics of photoinactivation. Additionally, these strains were grown at two light intensities to characterize their long-term resistance to photoinactivation. The orange strain 2.D37291S and yellow strain 1.C2y had the highest carotene contents and were the most light resistant in the kinetics and growth experiments. The pink strains 2.C425, AB278a-1, and 1.C421 accumulated cytochrome c as well as carotenes. These strains were slightly more photosensitive than the yellow or orange strains in the kinetics experiment but were much more sensitive in the growth experiment. The phytoene-containing white strain 2.C419, which contains a small amount of cytochrome c, had a high level of resistance to light in the kinetics and growth experiments. The carotene-less strains were most sensitive in the kinetics experiment but not in the growth experiment. The overall photosensitivity ofU. violacea strains is related to the carotene and cytochrome c contents.     

Drug resistance in Salmonellae isolated at Chandigarh (India) during 1972–1978

A total of 1316 strains of Salmonella belonging to 20 serotypes isolated at P.G.I. Chandigarh (India) were tested for drug resistance. Drug resistance was noticed in 494 (38.3%) of the strains; 194 (14.8%) of these strains were resistant to one drug, while 300 (23.5%) had multiple drug resistance. All isolated strains were sensitive to gentamicin, furazolidone and nalidixic acid.Resistance to streptomycin was observed in 233 (17.7%), chloramphenicol 197 (14,9%), tetracycline 293 (22.3%), ampicillin 428 (32.5%), kanamycin 206 (15.7%), neomycin 206 (15.7%) and sulphadiazine 215 (19.9%).Multiple drug resistance was most common in S. bareilly, S. typhimurium and S. anatum serotypes. Increase in incidence of drug resistance in Salmonellae has been noticed during 1972–1978.     

Experimental criteria for interpretation of bacterial sensitivity to dioxidine determined by diffusion from the disks

Antibacterial activity of dioxidine against aerobic and facultative anaerobic organisms under conditions of anaerobiosis i. e. conditions really observed for example in abscess cavities or necrotic tissues is 30 to 100 times as high as that under aerobic conditions. There is a relationship between sensitivity of bacteria to dioxidine under aerobic and anaerobic conditions which is expressed by the regression equation. Therefore, comparison of the MICs determined under anaerobic conditions with the growth inhibition zones formed by disks with the drug under aerobic conditions is possible. The MIC of dioxidine was determined under anaerobic conditions for 179 clinical strains of aerobic and facultative anaerobic bacteria and the growth inhibition zones of the same bacteria under aerobic conditions were evaluated with the use of disks containing 100, 75, 50, 25, 20, and 15 micrograms of the drug. The border line. MIC differentiating between resistant and sensitive strains was chosen to be equal to 4 micrograms/ml. Differentiation of the strains into sensitive and resistant ones by the values of the growth inhibition zones was performed with the method of error minimization described by C. Metzler and R. De Haan in 1974. Disks containing 25 micrograms of the drug allowed one to differentiate the strains under aerobic conditions into sensitive and resistant ones: the growth inhibition zones greater than 11 mm corresponded to the sensitive strains (the MIC smaller than 4 micrograms/ml) and the growth inhibition zones smaller than 11 mm corresponded to the resistant strains (the MIC greater than 4 micrograms/ml).     

Physiological characteristics ofClostridium bifermentans selectively isolated from California desert tortoise

A selective agar medium containing cycloserine (250 mg/L), sulfamethoxazole (76 mg/L), and trimethoprim (4 mg/L) was used for isolation ofClostridium bifermentans from the intestinal contents of California desert tortoise. Typical lecithinase positive colonies that appeared on the plates, were biochemically characterized with the API 20A System and a conventional procedure. The susceptibility of the isolates to 12 antimicrobial agents was determined by the broth microtitration technique using the ANA MIC System.C. bifermentans strains were shown to be highly susceptible to cefoxitin, cefotetan, ceftizoxime, cefotaxime, chloramphenicol, clindamycin, penicillin, metronidazole, piperacillin, ticarcillin, and mezlocillin. Less than 10% of the isolates exhibited resistance to tetracycline. All strains were found to be highly resistant to cycloserine, sulfamethoxazole, and trimethoprim. The GLC analysis of the culture supernatants for volatile fatty acids revealed the presence of formic, acetic, isobutyric, butyric, isovaleric, and isocaproic acids.     

Postantibiotic effects of imipenem and enoxacin againstS. typhimurium andS. enteritidis and the influence on their surface hydrophobicity

The influence of the postantibiotic effect (PAE) and the postantibiotic sub-MICs effect (PA SME) of imipenem and enoxacin on the surface hydrophobicity ofS. typhimurium andS. enteritidis strains were studied by evaluating Congo red binding and the aggregation in molar solutions of ammonium sulfate (SAT). A PAE was induced by 2× and 4× MIC of antibiotics tested for 0.5 h. Suprainhibitory concentrations of imipenem againstS. typhimurium induced a short PAE (0.3–0.6 h) compared toS. enteritidis (6.0–9.7 h). Suprasubinhibitory concentrations of imipenem did not allow a regrowth ofS. enteritidis. Similar results were also found for enoxacin. Evaluation of surface hydrophobic properties of the salmonellas after affecting both PAEs and PA SMEs has shown that imipenem at concentrations 4×MIC and 4×MIC+0.3×MIC partially influenced the hydrophobicity ofS. typhimurium. S. enteritidis was more susceptible toward both antibiotics tested.     

Use of paraffin bait technique in the isolation of Nocardia asteroides from sputum

Summary The use of paraffin bait technique in the isolation ofNocardia asteroides has been tested in 241 samples of sputa obtained from 235 cases of respiratory diseases.N. asteroides was recovered on 6 occasions from sputum of a patient using the paraffin bait technique. On the other hand cultures of sputa from the same patient on routine agar media such as Sabouraud's agar and Lowenstein Jensen medium yielded only one isolated of the pathogen.This work forms a part of the Ph. D. thesis of P.V.K. submitted to the University of Delhi.     

Frequency of resistance to tetracycline and chloramphenicol amongSalmonella strains isolated in the Netherlands in 1962

Out of 12,472 strains ofSalmonella isolated in the Netherlands in 1962, 1365, or 10.94%, were found to be resistant to tetracycline or chloramphenicol or to both. Compared with the findings of the preceding years (1958/59:2.08%, 1960:1.29%, 1961:3.96%) this is a considerable increase. Of these 1365 strains, 1285, or 94.1%, were resistant to tetracycline and 46, or 3.4%, were resistant to chloramphenicol. The remaining 34 strains, or 2.5%, were resistant to both drugs.Among allSalmonella strains isolated in 1962, 5517 belonged to the speciesS. typhi murium. Of these, 1203 or 21.8%, were resistant to tetracycline. The resistance rates of strains originating from human patients, calves, pigs, other animals and other materials were 24.4%, 37.1%, 15.0%, 8.0% and 5.7% respectively.Factors which may possibly have contributed to the greatly increased frequency of drug resistance inS. typhi murium are: (1) the rapid spread of the use of tetracycline for therapeutic, prophylactic and nutritive purposes, and (2) the possibility of an episome-mediated transfer of drug resistance from relatively harmless intestinal bacteria, such asE. coli, toS. typhi murium.     

Clinical isolates of <Emphasis Type="Italic">Nocardia brasiliensis</Emphasis> from Japan exhibit variable susceptibility to the antibiotic imipenem

Clinical isolates of Nocardia brasiliensis from Japan were classified into two groups based on their susceptibility to the carbapenem antibiotic, imipenem (IPM). Of 33 strains tested, 10 belonged to an IPM susceptible group, with MIC of from 0.25 to 2 εg/ml and a MIC₈₀ value of 1.5 εg/ml for this antibiotic. The remaining 23 strains belonged to an IPMresistant group with MIC and MIC₈₀ values of 8–16 εg/ml and >16 εg/ml, respectively. The type strain of N. brasiliensis belonged to this resistant group. Analysis of 16S rDNA genes sequences showed that the IPM susceptible group had characteristic single nucleotide substitutions at positions 103 (T), 381 (A), and 456 (A), in contrast to the IPM resistant group. This grouping, however, was not associated with their clinical manifestation.