Biofilm formation and antifungal susceptibility of Trichosporon asahii isolates from Mexican patients

Background

Trichosporon asahii is a yeast-like fungus that has recently gained importance as a cause of opportunistic systemic infections. The pathogenicity and virulence factors of T. asahii remain largely unknown. Because of the association between invasive infections and the use of catheters and related devices, the ability of the microorganism to adhere and form biofilms may play an important role in the pathogenicity during a trichosporonosis.

Activity of antifungal agents alone and in combination against echinocandin-susceptible and -resistant Candida parapsilosis strains

Background

Candida parapsilosis may acquire resistance to echinocandins, a fact that prompts the search for new therapeutic options.

棘白菌素对氟康唑耐药的念珠菌体外药物敏感性的研究

目的评价3种棘白菌素类药物（卡泊芬净、米卡芬净、阿尼多芬净）体外对氟康唑耐药念珠菌的药物敏感性。方法采用微量液体稀释法和琼脂稀释法测定最小抑制浓度（MIC）。结果微量液体稀释法：59株耐药白念珠菌3种药物MIC50均为0.06μg/mL,米卡芬净、阿尼多芬净的MIC范围均为0.015～0.125μg/mL,卡泊芬净为0.015～0.25μg/mL;8株耐药光滑念珠菌MIC值均为0.063μg/mL。琼脂稀释法：59株耐药白念珠菌和8株耐药光滑念珠菌3种药物MIC值均为0.063μg/mL。结论3种棘白菌素类药物可能具有治疗氟康唑耐药的念珠菌感染的临床价值。     

Antimicrobial susceptibility and genetic relatedness of bovine Stenotrophomonas maltophilia isolates from a mastitis outbreak

Aims: To evaluate the antimicrobial susceptibility and genetic relatedness of 11 Stenotrophomonas maltophilia isolates from an outbreak of bovine clinical mastitis in one herd and two isolates from two separate mastitis cases in two other herds. Methods and Results: Thirteen S. maltophilia isolates were obtained from milk samples from 11 cows from three dairy herds in Japan during 2008. We tested their susceptibility to 14 antimicrobials by broth microdilution and identified their genotypes by enterobacterial repetitive intergenic consensus 2 (ERIC2)‐PCR. Every cow had acute mild mastitis (slightly watery foremilk with flakes) without systemic symptoms and all resolved within 3–5 weeks of diagnosis. Eleven of the 13 isolates derived from nine cows in one herd over a 7‐month period exhibited a closely related ERIC2 type (A). The remaining two isolates derived from two cows from two other herds exhibited two distinct ERIC2 types (B and C). Most of the 13 isolates exhibited susceptibility to trimethoprim‐sulfamethoxazole, chloramphenicol, minocycline and levofloxacin; however, they were resistant to four β‐lactams, kanamycin, gentamicin and oxytetracycline. They were intermediate to enrofloxacin. Conclusions: Eleven closely related S. maltophilia isolates were involved in a herd outbreak of mastitis to some extent. Bovine S. maltophilia isolates exhibited resistance to many classes of antimicrobials. Significance and impact of study: This is a rare report of a herd outbreak of bovine mastitis involving closely related S. maltophilia isolates.     

Species distribution and antifungal susceptibility among clinical isolates of Candida parapsilosis complex from India

Background

Candida parapsilosis is recognized as a species complex: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis are three distinct but closely related species.

Molecular typing and antifungal susceptibility of clinical sequential isolates of Cryptococcus neoformans from Sao Paulo State, Brazil

The antifungal susceptibility profiles and the genetic variability of 83 sequential clinical isolates of Cryptococcus neoformans, including four Cryptococcus gattii isolates, obtained from 38 Sao Paulo AIDS patients with cryptococcal meningitis were assessed by electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis. The majority of the Cryptococcus neoformans isolates were highly susceptible to amphotericin B and fluconazole. Twenty percent of the minimum inhibitory concentration values for amphotericin B varied from 0.5 to 1 micro g mL(-1). For fluconazole, 22% occurred in the range 8-16 mug mL(-1). Sequential isolates from nine patients showed a trend towards lower susceptibility to fluconazole, flucytosine, itraconazole and amphotericin B. The results of molecular typing by electrophoretic karyotyping and RAPD analysis showed the presence of 22 electrophoretic karyotypes (EK) and 15 RAPD profiles that were highly correlated. Our results provided evidence for the occurrence of genetic changes in some strains associated with microevolution during the course of infection. We also observed both microevolution and simultaneous coinfection with two distinct Cryptococcus neoformans strains in one patient. In some patients, we found changed EK- and RAPD patterns in association with increased MIC values.     

Phenotypic and genotypic profile of clinical and animal multidrug‐resistant Salmonella enterica isolates from Mexico

Aims

The objective of this study was to obtain a phenotypic and genotypic profile of Salmonella enterica including multidrug‐resistant (MDR) isolates from food‐producing animals and clinical isolates, as well as their genetic relatedness in two different States of Mexico (Jalisco and State of Mexico).

Methods and Results

A total of 243 isolates were evaluated in terms of antimicrobial resistance (AMR) and related genes through a disk diffusion method and PCR respectively; we found 16 MDR isolates, all of them harbouring the bla_CMY gene but not qnr genes, these isolates represent less than 10% of the collection. The pulsed‐field gel electrophoresis revealed a higher genotypic similitude within isolates of State of Mexico than Jalisco.

Conclusions

A low percentage of Salmonella isolates were resistant to relevant antibiotics in human health, nevertheless, the AMR and involved genes were similar despite the different serovars and origin of the isolates.

Significance and Impact of the Study

This investigation provided an insight of the current status of AMR of Salmonella isolates in two States of Mexico and pinpoint the genes involved in AMR and their epidemiological relationship, the information could help to determine an adequate therapy in human and veterinary medicine.     

耐氟康唑念珠菌和耐伊曲康唑烟曲霉对泊沙康唑的敏感性测定

目的：测定耐氟康唑念珠菌和耐伊曲康唑烟曲霉临床分离株对泊沙康唑的敏感性。方法参照美国临床实验室标准化研究所制定的 M27-A3和 M38-A2方案,测定从临床获得的11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉对泊沙康唑的 MIC 值。结果对于氟康唑耐药的念珠菌,泊沙康唑的 MIC 范围是0.125-1μg/ mL。对于伊曲康唑耐药烟曲霉,泊沙康唑的 MIC 范围是0.06-0.5μg/ mL。结论11株耐氟康唑的念珠菌和3株耐伊曲康唑烟曲霉均对泊沙康唑有效。     

Comparison of DNA-based typing methods to assess genetic diversity and relatedness among Candida albicans clinical isolates

Three serial isolates of Candida albicans were obtained from each of five HIV infected patients with recurrent oropharyngeal candidiasis from the same geographical area. Isolates from one patient remained susceptible to fluconazole whereas serial isolates from the other four patients showed decreasing susceptibilities to the drug. Strain identity was investigated by pulse-field gel electrophoretic (PFGE) separation of chromosomes, restriction fragment length polymorphism (RFLP) of chromosomal DNA, Southern blot analysis with the moderately repetitive probe Ca3 of the materials present in the RFLP gels after transfer to nylon membranes, and random amplification of polymorphic DNA (RAPD). All techniques were able to group isolates obtained from the same patient. Techniques resulting in more complex banding profiles exhibited increased discriminatory power allowing detection of strain variants. Methods resulting in less complex banding patterns, especially Southern hybridization of SfiI digested chromosomal DNA with the moderately repetitive probe Ca3, were more helpful to determine isogenicity among isolates obtained from the same patient. The combination of results from methods with high discriminatory power (to maximize detection of strain variants) and methods resulting in less complex banding patterns (to allow determination of isogenic isolates) should facilitate the delineation of the epidemiology of C. albicans infection.     

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background

Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available.

Aim

This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates.

Methods

A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein-Jensen medium was used as reference test.

Results

Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8-14 days.

Conclusion

Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.     

Electrophoretic karyotyping and antifungal susceptibility patterns of Candida parapsilosis clinical isolates causing deep and superficial fungal infections

Forty-six isolates of Candida parapsilosis, each from a single patient, were collected from July 1993 through March 1999 at the University of Ancona Hospitals and Clinics. Twenty-eight strains were isolated from superficial lesioned sites, including skin, nails and other sources while 18 strains were isolated from blood. The isolates were typed by electrophoretickaryotyping (EK) and tested for their susceptibility to fluconazole (FLC),itraconazole (ITC), flucytosine (5-FC), and amphotericin B (AMB). Ourdata confirmed that EK is a useful technique for DNA typing of isolates ofCandida parapsilosis and showed that the source of isolation is notassociated with a given DNA type. Although strains belonging to this speciesof Candida are susceptible to the most common antifungals, including the triazoles, the degree of ITC susceptibility was dose dependent (MIC rangingfrom 0.25–0.5 μg/ml) for 98% of the isolates. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro susceptibility to 9 antifungal agents of 14 strains of Zygomycetes isolated from clinical specimens

Fourteen clinical isolates of Zygomycetes were tested for their in vitro susceptibility to nine antifungal agents. Susceptibility assessment was performed using a microtiter broth dilution method. Synthetic broth with YNB and glucose was used for 5-fluorocytosine and BHI broth for all the other antimycotics. Amphotericin B exhibited the strongest activity against all isolates tested. MIC values of other two polyenes — nystatin and pimaricin — ranged within the susceptibility limits, with a little pronounced higher activity of pimaricin. The isolates of the genusAbsidia andSyncephalastrum were well sensitive to all antimycotics with the exception of 5-fluorocytosine and naftifine. A very weak or zero growth inhibitory effect against all members of the generaMucor andRhizopus was found in azoles, 5-fluorocytosine and naftifine.     

In vitro activity of the lipopeptide PAL-Lys-Lys-NH2, alone and in combination with antifungal agents, against clinical isolates of Candida spp

Candida albicans is known to be the organism most often associated with serious fungal infection, but other Candida spp. are emerging as clinical pathogens associated with opportunistic infections. Among antimycotic treatments, increasing attention is currently given to anti-infective drugs based upon naturally occurring peptides, such as the short lipopeptide palmitoyl PAL-Lys-Lys-NH2 (PAL). The aim of this study is to evaluate the activity of this peptide compared to the traditional antifungal agents Fluconazole (FLU), amphotericin B (AMB) and caspofungin (CAS) on Candida spp. 24 clinical isolates of Candida spp. were tested against PAL_, FLU, AMB and CAS using in vitro susceptibility tests, time killing and checkerboard assay. All of the drugs studied showed good activity against clinical isolates of candida; in particular CAS and AMB which have MICs value lower than PAL and FLU. Moreover we observed synergistic interactions for PAL/FLU (81.25%), PAL/AMB (75%) and particularly for PAL/CAS (87.5). We think that our results are interesting since synergy between PAL and CAS might be useful in clinic trails to treat invasive fungal infections.     

Serovar distribution and antimicrobial susceptibility of swine Salmonella isolates from clinically ill pigs in diagnostic submissions from Indiana in the United States

Aims:  To determine serovar distribution and levels of antimicrobial susceptibility of Salmonella isolated from clinically ill pigs in diagnostic submissions.
Methods and Results:  A total of 197 Salmonella isolates were obtained by the Indiana Animal Disease Diagnostic Laboratory from 2003 to 2005. Minimal inhibitory concentrations (MICs) were determined using the standard microbroth dilution method. The top four serovars identified were Salm. enterica serovar Typhimurium variant Copenhagen, Salm . Derby, Salm . Choleraesuis var. Kunzendorf and Salm . Typhimurium. All isolates were susceptible to the fluoroquinolones tested except that eight isolates were intermediate to difloxacin. The isolates showed a low prevalence of resistance to trimethoprim/sulphadiazine (Sxt), gentamicin (G), ceftiofur (Cf) and cephalothin (Cp) with low MIC₅₀ value of ≤0·5, 0·5, 1 and 4  μ g ml⁻¹, respectively. They showed a high prevalence of resistance to tetracycline (T; 83·8%), and a moderate prevalence to ampicillin (55·8%), spectinomycin (42·6%), ticarcillin (41·6%) and florfenicol (41·1%). There were more isolates of Salm . Typhimurium, including var. Copenhagen and Salm . Agona, that possessed multiple antimicrobial resistance to amoxicillin/clavulanic acid, ampicillin, ceftiofur and cephalothin (AxApCfCp) than the other serovars.
Conclusions:  The swine Salmonella isolates were susceptible to the fluoroquinolones, Sxt, G, Cf and Cp, but resistant to T.
Significance and Impact of the Study:  These findings provided useful information regarding antimicrobial susceptibility and resistance in dealing with clinical salmonellosis in pig herds.     

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.     

Leishmania donovani: effect of verapamil on in vitro susceptibility of promastigote and amastigote stages of Indian clinical isolates to sodium stibogluconate

Although pentavalent antimonials are the first-line drug for treatment of visceral leishmaniasis all over the world, yet, in India, increasing number of patients are being reported to be unresponsive to sodium stibogluconate. Verapamil, a calcium channel blocker, affects drug uptake by preventing its efflux and thereby accumulation within the cell. In the present study, effect of verapamil on in vitro susceptibility of both promastigote and amastigote stages of 15 clinical isolates and standard strain of Leishmania donovani to sodium stibogluconate was evaluated by detection of acid phosphatase. Amastigotes were found more susceptible to sodium stibogluconate than the promastigotes (p<0.05) and in the presence of verapamil, IC(50) value of sodium stibogluconate was reduced only for those isolates, which had a higher IC(50). Verapamil alone did not have any effect on the parasites. The results indicate that amastigotes are more susceptible to sodium stibogluconate than promastigotes and verapamil can reverse the in vitro drug resistance of L. donovani clinical isolates to sodium stibogluconate.     

Field evaluation of anthelmintic drug sensitivity using in vitro egg hatch and larval motility assays with Necator americanus recovered from human clinical isolates

A field-applicable assay for testing anthelmintic sensitivity is required to monitor for anthelmintic resistance. We undertook a study to evaluate the ability of three in vitro assay systems to define drug sensitivity of clinical isolates of the human hookworm parasite Necator americanus recovered from children resident in a village in Madang Province, Papua New Guinea. The assays entailed observation of drug effects on egg hatch (EHA), larval development (LDA), and motility of infective stage larvae (LMA). The egg hatch assay proved the best method for assessing the response to benzimidazole anthelmintics, while the larval motility assay was suitable for assessing the response to ivermectin. The performance of the larval development assay was unsatisfactory on account of interference caused by contaminating bacteria. A simple protocol was developed whereby stool samples were subdivided and used for immediate egg recovery, as well as for faecal culture, in order to provide eggs and infective larvae, respectively, for use in the egg hatch assay and larval motility assay systems. While the assays proved effective in quantifying drug sensitivity in larvae of the drug-susceptible hookworms examined in this study, their ability to indicate drug resistance in larval or adult hookworms remains to be determined.