High MICs Research Articles

Evaluation of modified carbapenem inactivation method for suspected carbapenemase among Enterobacteriaceae clinical isolates.

Modified carbapenem inactivation method (mCIM) testing was currently recommended by Clinical and Laboratory Standards Institute (CLSI) for detection of carbapenemase among Enterobacteriaceae clinical isolates. In this study, a panel of 145 clinical strains were collected for evaluating the mCIM for detection of carbapenemase. Antimicrobial susceptibility testing were performed by microbroth dilution and the results were interpreted according to CLSI guidelines. All strains were resistant to ertapenem with high MIC50 and MIC90 (64 mg/L –>128 mg/L). For blaNDM-1-positive or blaOXA-232-positive strains, the zone of inhibition of meropenem were all 6 mm despite the incubation time of 6 h, 18 h or 24 h. For 6 h, the zone of meropenem inhibition for most of carbapenemase-positive isolates were meet the positive criteria 6–15 mm. However, for carbapenemase-negative isolates, the zone of meropenem inhibition were 16–18 mm after 6 h incubation which should be considered indeterminate for standard incubating time such as 18 h or 24 h. After incubating for 18 h or 24 h, the zone of meropenem inhibition were 22–25 mm for carbapenemase-negative isolates and meet the negative criteria. Our study indicate mCIM is a simple and effective method to identify the carbapenemases producers among Enterobacteriaceae clinical isolates.

Airway persistence by the emerging multi‐azole‐resistant Rasamsonia argillacea complex in cystic fibrosis

Infections caused by Rasamsonia argillacea complex have been reported in various clinical settings. Cystic fibrosis (CF) is one of the main underlying conditions. An observational cohort study of CF patients with Rasamsonia in respiratory samples was conducted. Eight isolates from 6 patients were identified as R.argillacea complex and tested for antifungal susceptibility. All isolates had high MICs to voriconazole and posaconazole and low MECs to echinocandins. Four patients experienced lung function decline in the year preceding first Rasamsonia isolation. This continued in the year following first isolation in 3 out of 4 cases. Antifungal therapy was initiated in 2 patients, to which only one exhibited a clinical response. Three out of 6 patients died within 3years of isolating Rasamsonia. Genotyping suggests that similar genotypes of Rasamsonia can persist in CF airways. Consistent with other fungi in CF, the clinical impact of airway colonisation by Rasamsonia is variable. In certain patients, Rasamsonia may be able to drive clinical decline. In others, though a clear impact on lung function may be difficult to determine, the appearance of Rasamsonia acts as a marker of disease severity. In others it does not appear to have an obvious clinical impact on disease progression.

Distribution of biocide resistant genes and biocides susceptibility in multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii - A first report from the Kingdom of Saudi Arabia.

The aim of this study was to determine the frequency of biocide resistant genes, qacA, qacE and cepA in multidrug resistant (MDR) bacteria: Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii and to correlate the presence or absence of resistant genes with biocides susceptibility. The study included 44 MDR K. pneumoniae, P. aeruginosa and A. baumannii microorganisms. The bacteria were screened for the presence of biocide resistant genes by the polymerase chain reaction (PCR) method. The test organisms were isolated from various clinical specimens in the Qassim region, Saudi Arabia. The in vitro susceptibility tests of the three biocides (benzalkonium chloride, cetrimide and chlorhexidine gluconate) were studied against the test isolates by broth microdilution method following Clinical and Laboratory Standards Institute guidelines. With the distribution of biocide resistant genes in K. pneumoniae, all 9 isolates (100%) possessed cepA; 4 (44.4%) and 1 (11.1%) isolate contained qacA and qacE genes respectively. Among 24 isolates of A. baumannii tested, cepA, qacA and qacE genes were found in 54.2%, 16.7% and 33.3% of isolates respectively. Among 11 P. aeruginosa isolates, 63.6% contained cepA gene, 18.2% contained qacE genes, and none of the isolates harboured qacA gene. There was no significant correlation between presence or absence of biocide resistant genes and high MIC values of the test isolates (p≥0.2). Our observations imply that there was no significant correlation between presence or absence of biocide resistant genes and MICs observed in MDR K. pneumoniae, P. aeruginosa and A. baumannii. Further studies are required to find to confirm the trend of reduced susceptibility to biocides of problematic nosocomial pathogens.

Pharmacodynamics of Voriconazole for Invasive Pulmonary Scedosporiosis

Scedosporium apiospermum is a medically important fungal pathogen that causes a wide range of infections in humans. There are relatively few antifungal agents that are active against Scedosporium spp. Little is known about the pharmacodynamics of voriconazole against Scedosporium Both static and dynamic in vitro models of invasive scedosporiosis were developed. Monoclonal antibodies that target a soluble cell wall antigen secreted by Scedosporium apiospermum were used to describe the pharmacodynamics of voriconazole. Mathematical pharmacokinetic-pharmacodynamic models were fitted to the data to estimate the drug exposure required to suppress the release of fungal antigen. The experimental results were bridged to humans using Monte Carlo simulation. All 3 strains of S. apiospermum tested invaded through the cellular bilayer of the in vitro models and liberated antigen. There was a concentration-dependent decline in the amount of antigen, with near maximal antifungal activity against all 3 strains being achieved with voriconazole at 10 mg/liter. Similarly, there was a drug exposure-dependent decline in the amount of circulating antigen in the dynamic model and complete suppression of antigen, with an area under the concentration-time curve (AUC) of approximately 80 mg · h/liter. A regression of the AUC/MIC versus the area under the antigen-time curve showed that a near maximal effect was obtained with an AUC/MIC of approximately 100. Monte Carlo simulation suggested that only isolates with an MIC of 0.5 mg/liter enabled pharmacodynamic targets to be achieved with a standard regimen of voriconazole. Isolates with higher MICs may need drug exposure targets higher than those currently recommended for other fungi.

Mutation in the Squalene Epoxidase Gene of Trichophyton interdigitale and Trichophyton rubrum Associated with Allylamine Resistance.

Dermatophytosis, the commonest superficial fungal infection, has gained recent attention due to its change of epidemiology and treatment failures. Despite the availability of several agents effective against dermatophytes, the incidences of chronic infection, reinfection, and treatment failures are on the rise. Trichophyton rubrum and Trichophyton interdigitale are the two species most frequently identified among clinical isolates in India. Consecutive patients (n = 195) with suspected dermatophytosis during the second half of 2014 were included in this study. Patients were categorized into relapse and new cases according to standard definitions. Antifungal susceptibility testing of the isolated Trichophyton species (n = 127) was carried out with 12 antifungal agents: fluconazole, voriconazole, itraconazole, ketoconazole, sertaconazole, clotrimazole, terbinafine, naftifine, amorolfine, ciclopirox olamine, griseofulvin, and luliconazole. The squalene epoxidase gene was evaluated for mutation (if any) in 15 T. interdigitale and 5 T. rubrum isolates exhibiting high MICs for terbinafine. A T1189C mutation was observed in four T. interdigitale and two T. rubrum isolates. This transition leads to the change of phenylalanine to leucine in the 397th position of the squalene epoxidase enzyme. In homology modeling the mutant residue was smaller than the wild type and positioned in the dominant site of squalene epoxidase during drug interaction, which may lead to a failure to block the ergosterol biosynthesis pathway by the antifungal drug.

Cationic amphiphilic bolaamphiphile-based delivery of antisense oligonucleotides provides a potentially microbiome sparing treatment for C. difficile.

Conventional antibiotics for C. difficile infection (CDI) have mechanisms of action without organismal specificity, potentially perpetuating the dysbiosis contributing to CDI, making antisense approaches an attractive alternative. Here, three (APDE-8, CODE-9, CYDE-21) novel cationic amphiphilic bolaamphiphiles (CABs) were synthesized and tested for their ability to form nano-sized vesicles or vesicle-like aggregates (CABVs) which were characterized based on their physiochemical properties, their antibacterial activities, and their toxicity toward colonocyte (Caco-2) cell cultures. The antibacterial activity of empty CABVs were tested against cultures of E. coli, B. fragilis and E. faecalis, and against C. difficile by “loading” CABVs with 25-mer antisense oligonucleotides (ASO) targeting dnaE. Our results demonstrate that empty CABVs have minimal colonocyte toxicity until concentrations of 71 μM, with CODE-9 demonstrating the least toxicity. Empty CABVs had little effect on C. difficile growth in culture (MIC90 ≥160 μM). While APDE-8 and CODE-9 nanocomplexes demonstrated high MIC90 against C. difficile cultures (>300 μM), CYDE-21 nanocomplexes demonstrated MIC90 at CABV concentrations of 19 μM. Empty CABVs formed from APDE-8 and CODE-9 had virtually no effect on E. coli, B. fragilis and E. faecalis across all tested concentrations, while empty CYDE-21 demonstrated MIC90 of >160 μM against E. coli and >40μM against B. fragilis and E. faecalis.Empty CABVs have limited antibacterial activity and they can deliver an amount of ASO effective against C. difficile at CABV concentrations associated with limited colonocyte toxicity, while sparing other bacteria. With further refinement, antisense therapies for CDI may become a viable alternative to conventional antibiotic treatment.

Bacteroides fragilis: A whole MALDI-based workflow from identification to confirmation of carbapenemase production for routine laboratories

Bacteroides fragilis is a frequent anaerobic pathogen and can cause severe infections. Resistance to carbapenems, associated with the cfiA gene encoded carbapenemase, represents an emerging problem. To date, no rapid methods are available to detect and confirm this resistance mechanism in routine laboratories, and the missed recognition of carbapenemase-producing strains can lead to therapeutic failures. In this study we have investigated a whole MALDI-TOF MS-based workflow to detect carbapenemase-producing B. fragilis, using the largest set of B. fragilis clinical isolates ever tested. The presence of the cfiA gene was predicted by MALDI subtyping into Division I (cfiA-negative) or Division II (cfiA-positive). The carbapenemase activity in cfiA-positive strains was confirmed by a MALDI-TOF MS imipenem hydrolysis assay (MBT STAR-Carba, Bruker Daltonik, Germany), that was further used for a characterization of the strains in terms of cfiA expression level.The validity of MALDI subtyping was verified by PCR for the cfiA gene, while results of MALDI hydrolysis assay were compared to conventional methods for susceptibility testing and carbapenemase detection (Carba-NP and disk diffusion synergy test). A genetic analysis of the IS elements upstream cfiA was performed, for the evaluations regarding the expression level of cfiA. A total of 5300 B. fragilis isolates (406 from Bologna, Italy, and 4894 from Dortmund, Germany) were identified and subtyped by MALDI-TOF MS, yielding 41/406 (10.1%) strains from Bologna and 374/4894 (7.6%) from Dortmund to belong to Division II. Molecular verification by PCR for the cfiA gene on a subset of strains confirmed the MALDI typing results in all cases (sensitivity and specificity of 100%). MBT STAR-Carba assay detected the carbapenemase activity in all of the 70 cfiA-carrying strains tested. Moreover, it allowed distinct separation into slow (59) and fast (11) imipenem hydrolyzers corresponding to cfiA expression levels as well as to low or high MICs for carbapenems, respectively. Among the 11 cfiA-positive strains with high carbapenem MIC, only 7 harboured IS elements upstream the carbapenemase gene showing low expression level as well.The MALDI-TOF MS-based workflow was superior to the currently available phenotypic methods for carbapenemase detection as it proved to be more sensitive and accurate than Carba NP and disk diffusion synergy test. The whole MALDI-TOF MS-based workflow allows an accurate identification of B. fragilis clinical strains with reliable classification into Division I/II, and confirmation of the carbapenemase-production, together with estimation of carbapenemase activity, within less than 2 h. This may be of particular interest for early therapeutical decisions in life-threatening infections.

Surveillance for azoles resistance in Aspergillus spp. highlights a high number of amphotericin B-resistant isolates.

Aspergillus spp. are the most common invasive mould infection and are responsible for high mortality. Aspergillus fumigatus is currently of interest because resistance to azole antifungals has emerged. The Campinas University Hospital (HC-UNICAMP) receives high-risk patients susceptible to opportunistic infections but there have been no reports of resistant A.fumigatus. This study aimed to assess the susceptibility profile of Aspergillus isolates, specifically looking for azole resistance. ITS and β-tubulin DNA sequencing was performed on 228 sequential clinical isolates. Broth microdilution susceptibility testing was performed for all isolates. A.fumigatus represented 74% of the isolates followed by Aspergillus flavus (12%). Nine A.fumigatus isolates from 9 different patients showed high MIC values to at least 1 azole, but cyp51A polymorphisms were detected in only 6 isolates and none correlated with known resistance mutations. The most troubling observation was that the minimum inhibitory concentration for amphotericin B was elevated (≥2mgL-1 ) in 87% of patients with A.flavus isolates and 43% with Aspergillusfumigatus isolates. Given that amphotericin B is used to treat azole-resistant infections, these data highlight the need for continuous surveillance in Aspergillus for all antifungal resistance to implement correct treatment strategies for the management of these pathogens.

In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

Investigation of Multiple Resistance Mechanisms in Voriconazole-Resistant Aspergillus flavus Clinical Isolates from a Chest Hospital Surveillance in Delhi, India.

Invasive and allergic infections by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in A. flavus impacts the management of aspergillosis, as azoles are used as the first-line and empirical therapy. We screened 120 molecularly confirmed A. flavus isolates obtained from respiratory and sinonasal specimens in a chest hospital in Delhi, India, for azole resistance using the CLSI broth microdilution (CLSI-BMD) method. Overall, 2.5% (n = 3/120) of A. flavus isolates had VRC MICs above epidemiological cutoff values (>1 μg/ml). The whole-genome sequence analysis of three non-wild-type (WT) A. flavus isolates with high VRC MICs showed polymorphisms in azole target genes (cyp51A, cyp51B, and cyp51C). Further, four novel substitutions (S196F, A324P, N423D, and V465M) encoded in the cyp51C gene were found in a single non-WT isolate which also exhibited overexpression of cyp51 (cyp51A, -B, and -C) genes and transporter genes, namely, MDR1, MDR2, atrF, and mfs1 The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effects on cyp51C drug binding. The substrate (drug) may not be able to bind to binding pocket due to changes in the pocket size or closing down or narrowing of cavities in drug entry channels. Notably, the remaining two VRC-resistant A. flavus isolates, including the one which had a pan-azole resistance phenotype (itraconazole and posaconazole), did not show upregulation of any of the analyzed target genes. These results suggest that multiple target genes and mechanisms could simultaneously contribute to azole resistance in A. flavus.

Prevalence of carbapenem resistance genes and corresponding MIC90 in Enterobacteriaceae at a tertiary care center in Lebanon.

The aim of this study was to correlate genes involved in carbapenem resistance to MIC levels among clinical ESBL and non-ESBL producing carbapenem resistant Enterobacteriaceae (CRE) isolates of Escherichia coli and Klebsiella pneumoniae. E. coli (n = 76) and K. pneumoniae (n = 54), collected between July 2008 and July 2014, were analyzed. The MICs were determined against ertapenem (ERT), imipenem (IMP) and meropenem (MER). PCR was performed on all 130 isolates to amplify the resistance and outer membrane proteins (OMPs) encoding genes: blaOXA-48, blaNDM-1, blaTEM-1, blaCTX-M-15, ompC and ompF. Sequencing was performed on selected isolates. The prevalence of blaOXA-48, blaNDM-1, blaTEM-1, and/or blaCTX-M-15 among E. coli isolates were 36%, 12%, 20% and 80%, respectively, while among K. pneumoniae they were 37%, 28%, 28% and 72%, respectively. K. pneumoniae isolates positive for any of these genes had an MIC90 > 32μg/mL against ERT, IMP and MER, while in E. coli isolates there was a variation in the MIC90 values. Porin impermeabilities were due to mutations in ompC and ompF genes in E. coli, and loss of ompC and ompF genes in K. pneumoniae, and increased MIC90 values. The presence of more than one carbapenem resistance encoding gene and/or ESBL encoding gene did not have an effect on the MIC90 value in K. pneumoniae isolates, while in E. coli it showed higher MIC90 values.

Antimicrobial susceptibility of Haemophilus parasuis isolates from Germany by use of a proposed standard method for harmonized testing

Haemophilus parasuis-related infections, especially among weaners are responsible for major economic losses on pig farms. A method for broth microdilution susceptibility testing of this fastidious organism has recently been developed, but the suitability of this method needs to be validated in a large collection of current field isolates. Using the proposed method, this study tested 123 H. parasuis isolates from different geographic regions in Germany (including five isolates from the Netherlands and Belgium) against a panel of 24 antimicrobial agents and antimicrobial combinations. The isolates were collected between 2013 and 2016. As there are no H. parasuis specific breakpoints available, the tested isolates could not be classified as susceptible, intermediate or resistant. Bi- or multi-modal distributions of minimum inhibitory concentration (MIC) values were observed for some antimicrobial agents (e.g. aminoglycosides, β-lactams, fluoroquinolones and tetracyclines), indicative of non-wild type populations of H. parasuis. Susceptibility testing revealed broad distributions of MIC values for various antimicrobials (e.g. neomycin, streptomycin, tetracycline, tiamulin, tilmicosin, trimethoprim/sulfamethoxazole and tulathromycin). The lowest MIC90 (i.e. the concentration at which 90% of isolates were inhibited) was obtained for cefotaxime (≤0.015 μg/ml), and the highest MIC90 (512 μg/ml) was obtained for streptomycin. This study tested a large set of current field isolates and included the most common serovars (serovars 4 and 5). The results point to the suitability of the broth microdilution susceptibility testing method proposed previously for determining H. parasuis MIC values. In addition, the study provides a reliable overview of the susceptibility status of H. parasuis at present in Germany.

Efficacy of different fungicides against the leaf spot of lettuce caused by Allophoma tropica

The efficacy of different fungicides, belonging to different chemical groups, was in vitro and in vivo tested in glasshouse trials against Allophoma tropica, the causal agent of a new leaf spot on lettuce. The in vitro trials showed that only a few fungicides were effective in inhibiting the mycelial growth of the four tested strains of A. tropica: the most effective fungicide, with the lowest ED50 and MIC values, was penconazole. Prochloraz, propiconazole, boscalid and fludioxonil showed low ED50 values, but high MIC values. The results from the in vivo trials showed that all the tested fungicides significantly reduced Phoma leaf spot incidence and severity, although differences were observed in the achieved disease pressure in the four trials. Generally, in the presence of an average disease severity in the untreated control, the best disease severity reduction (up to 90%) was provided 9 days after the application of fludioxonil, mancozeb, pyraclostrobin + boscalid, propiconazole and iprodione. Fludioxonil and iprodione showed a persistent effect (up to 90%) still being evident 12 days after the application. Generally, in the presence of a high disease severity in the untreated control, 9 days after the application of fludioxonil, mancozeb, iprodione and pyraclostrobin + boscalid provided a disease severity reduction up to 80%. Moreover, copper provided a disease severity reduction from 84 to 89%, applied as preventative treatment, resulting of special interest, particularly for crops grown under organic farming rules.

Vibrio parahaemolyticus and Vibrio vulnificus Recovered from Oysters during an Oyster Relay Study.

Vibrio parahaemolyticus and Vibrio vulnificus are naturally occurring estuarine bacteria and are the leading causes of seafood-associated infections and mortality in the United States. Though multiple-antibiotic-resistant V. parahaemolyticus and V. vulnificus strains have been reported, resistance patterns in vibrios are not as well documented as those of other foodborne bacterial pathogens. Salinity relaying (SR) is a postharvest processing (PHP) treatment to reduce the abundances of these pathogens in shellfish harvested during the warmer months. The purpose of this study was to evaluate the antimicrobial susceptibility (AMS), pathogenicity, and genetic profiles of V. parahaemolyticus and V. vulnificus recovered from oysters during an oyster relay study. Isolates (V. parahaemolyticus [n = 296] and V. vulnificus [n = 94]) were recovered from oysters before and during the 21-day relaying study to detect virulence genes (tdh and trh) and genes correlated with virulence (vcgC) using multiplex quantitative PCR (qPCR). AMS to 20 different antibiotics was investigated using microbroth dilution, and pulsed-field gel electrophoresis (PFGE) was used to study the genetic profiles of the isolates. Twenty percent of V. vulnificus isolates were vcgC+, while 1 and 2% of V. parahaemolyticus were tdh+ and trh+, respectively. More than 77% of the V. vulnificus isolates and 30% of the V. parahaemolyticus isolates were resistant to at least one antimicrobial. Forty-eight percent of V. vulnificus and 8% of V. parahaemolyticus isolates were resistant to two or more antimicrobials. All isolates demonstrated a high genetic diversity, even among those isolated from the same site and having a similar AMS profile. No significant effects of the relaying process on AMS, virulence genes, or PFGE profiles of V. vulnificus and V. parahaemolyticus were observed.IMPORTANCE Analysis of the antibiotic resistance profiles of V. vulnificus and V. parahaemolyticus isolated from oysters during this study indicated that more than 48% of V. vulnificus isolates were resistant to two or more antimicrobials, including those recommended by the CDC for treating Vibrio infections. Also, the V. parahaemolyticus isolates showed high MICs for some of the Vibrio infection treatment antibiotics. Monitoring of AMS profiles of this bacterium is important to ensure optimal treatment of infections and improve food safety. Our study showed no significant differences in the AMS profiles of V. vulnificus (P = 0.26) and V. parahaemolyticus (P = 0.23) isolated from the oysters collected before versus after relaying. This suggests that the salinity of the relaying sites did not affect the AMS profiles of the Vibrio isolates, although it did reduce the numbers of these bacteria in oysters (S. Parveen et al., J Food Sci 82:484-491, 2017, https://doi.org/10.1111/1750-3841.13584).

Comparative evaluation of sensititre yeast one panel against broth microdilution method for testing Candida auris and Candida haemulonii susceptibility: A pilot study

BACKGROUND: Candidal infection due to non-Candida albicans is on the rise and implicated to cause significant morbidity and mortality. Although empirical treatment can be provided, the sensitivity pattern varies drastically in different species of Candida. The conventional broth microdilution method for antifungal susceptibility is labour intensive. Data on antifungal susceptibility testing (AFST) by colorimetric yeast one sensititre method are available for many Candida species but not for Candida auris and Candida haemulonii. AIM: The utility of colorimetric yeast one plate was compared to conventional broth microdilution method (CLSI M27-A3 guidelines) for five isolates each of C. haemulonii and C. auris along with CLSI quality control strain Candida krusei ATCC 6258. MATERIALS AND METHODS: A total of 10 isolates of C. auris/haemulonii, confirmed by sugar assimilation and DNA sequencing were chosen. AFST was performed by conventional broth microdilution and colorimetric yeast one sensititre plates. STATISTICAL ANALYSIS: The analysis was performed by Carl Pearson's correlation coefficient. RESULTS: All isolates showed high MIC (>64 μg/mL) for Fluconazole by both methods. Strains of C. auris were found to have high MIC (>64 μg/mL) for Amphotericin B by conventional method. The degree of agreement between the two methods was 100%, 96.83% and 90.03% for Fluconazole, Posaconazole and Itraconazole, respectively, for the 10 isolates. The degree of agreement was 100%, 95.70% and 93.83% and 100%, 97.97% and 86.24% for Fluconazole, Posaconazole and Itraconazole for C. auris and C. haemulonii, respectively. CONCLUSION: AFST by colorimetric yeast one plate is less time-consuming, easy to perform and interpret, however further studies with a large number of isolates are required to test Echinocandins and Amphotericin B.

A Retrospective Analysis of Anaerobic Bacteria Isolated in 236 Cases of Pleural Empyema and their Prevalance of Antimicrobial Resistance in Turkey.

Parapneumonic effusions usually occur secondary to an infection and produce pus (empyema) that accumulates in the pleural space. We aimed to evaluate the prevalence of anerobes in patients with empyema and to assess their resistance patterns for seven antimicrobials. Pleural fluid specimens from 236 patients were inoculated on Schaedler agar. Anaerobic bacteria were identified via API 20 A. Susceptibility testing for penicillin, ampicillin + sulbactam, amoxicillin + clavulanate, cefoxitin, clindamycin, metronidazole, and imipenem were performed with the E-test. There were 118 anaerobic bacterial strains detected in 66 (27.9%) of the 236 specimens. Gram-positive anaerobic cocci were detected in 54.23% and the predominant cocci were 41 Peptostreptococcus spp, (34.75%) followed by 17 P. acnes (14.41%) and 6 C. tertium (5.08%). The Gram-negative anaerobes were B. fragilis (28, 23.73%), P. melaninogenica (8, 6.78%), P. intermedia (4, 3.39%), F. nucleatum (6, 5.08%), F. mortiferum (5, 4.24%), and P. asaccharolytica (3, 2.54%). All anaerobic strains were susceptible to ampicillin + sulbactam, amoxicillin + clavulanate, and imipenem. The highest MIC was found to be > 256 µg/mL for penicillin in B. fragilis strains, 128 µg/mL for cefoxitin in P. melaninogenica strains, 32 µg/mL for clindamycin and 64 µg/mL for metronidazole in P. acnes strains. Clindamycin resistance was detected in 46.6% B. fragilis, and 17.6% for P. acnes. Thirty-eight (32.2%) strains produced beta-lactamase. The use of antimicrobial agents for thoracic empyema should be based on the isolated pathogens and their resistance profiles. Clinicians should be aware of the wide diversity of anaerobic genera and species in cases of pleural empyema.

Protective Efficacy ofStreptococcus ThermophilusAgainst Acute Cadmium Toxicity in Mice.

Cadmium (Cd) is a highly toxic heavy metal, wide occupational and an environmental pollutant, affecting human health. Probiotics especially lactic acid bacteria (LAB) have the capacity to bind, remove and to decrease tissue cadmium levels. The objective was to evaluate the potency of Cd binding capacity, antioxidative properties of probiotic bacteria against cadmium in-vitro and its probable detoxification effect against Cd-induced toxicity in mice. To asses this objective, resistance against cadmium and antioxidative properties (via DPPH radical scavenging and inhibition of lipid peroxidation) were estimated for thirteen probiotic bacteria. Streptococcus thermophilus was selected among investigated bacteria as it had the highest MIC against cadmium and remarkable antioxidant activities for treatment of Cd toxicity in Swiss albino mice by preventive and therapeutic protocols. Blood cadmium levels, reduced glutathione (GSH), malondialdehyde (MDA) and histopathological changes in the liver of mice were estimated at 6, 24 and 48 h post to acute Cd exposure (oral dose with 50 mg/kg body weight). On exposure to Cd a significant increase in blood Cd, MDA and reducing in GSH levels were observed. S. thermophilus offered a significant protective effect against Cd toxicity by decreasing the cadmium levels in blood and attenuation alterations in the levels of GSH and MDA and improved hepatic histopathological changes caused by Cd toxicity. These results indicated the protective action of S. thermophilus against acute cadmium toxicity as well as their beneficial health effects and suggested its use as a safe and efficacious nutritional dietary supplement to reduce cadmium toxicity.

Candida infanticola and Candida spencermartinsiae yeasts: Possible emerging species in cancer patients

Opportunistic infections due to Candida species occur frequently in intensive care settings. We investigated the prevalence of Candida species among 65 clinical specimens obtained from 200 cancer patients by phenotypic and molecular (ITS sequencing and AFLP) methods. Among the 65 yeast isolates, Candida albicans was the most commonly isolated species (n = 34, 52.3%), whereas other Candida species comprised 47.7% (n = 31) and consisted of Candida glabrata (n = 14, 21.5%), Candida tropicalis (n = 5, 7.7%) and uncommon Candida species (n = 12, 18.5%) such as Candida pelliculosa (n = 3, 4.6%), Pichia kudriavzevii (= Candida krusei, n = 2, 3.1%), Candida orthopsilosis (n = 2, 3.1%), Candida parapsilosis (n = 1, 1.5%), Candida infanticola (n = 2, 3.1%), Candida spencermartinsiae (n = 1, 1.5%), and Kluyveromyces marxianus (=Candida kefyr, n = 1, 1.5%). Candida infanticola and Candida spencermartinsiae were recovered from oral lesions of cancer patients. Matrix assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) easily confirmed these isolates as less common Candida isolates (4.6%). The in vitro antifungal susceptibilities of C. spencermartinsiae and the two strains of C. infanticola were determined according to CLSI guidelines (M27-A3). MIC results among these isolates showed they were susceptible to isavuconazole, posaconazole and voriconazole, however, fluconazole and caspofungin had high MIC values. These Candida species that may occur more commonly in infections remain unnoticed using commonly used phenotypical methods in routine microbiology laboratories. MALDI-TOF MS proved to be a more fast and robust diagnostic technique for identification of the yeasts isolated from different clinical specimens of cancer patients.

En Route towards European Clinical Breakpoints for Veterinary Antimicrobial Susceptibility Testing: A Position Paper Explaining the VetCAST Approach.

VetCAST is the EUCAST sub-committee for Veterinary Antimicrobial Susceptibility Testing. Its remit is to define clinical breakpoints (CBPs) for antimicrobial drugs (AMDs) used in veterinary medicine in Europe. This position paper outlines the procedures and reviews scientific options to solve challenges for the determination of specific CBPs for animal species, drug substances and disease conditions. VetCAST will adopt EUCAST approaches: the initial step will be data assessment; then procedures for decisions on the CBP; and finally the release of recommendations for CBP implementation. The principal challenges anticipated by VetCAST are those associated with the differing modalities of AMD administration, including mass medication, specific long-acting product formulations or local administration. Specific challenges comprise mastitis treatment in dairy cattle, the range of species and within species breed considerations and several other variable factors not relevant to human medicine. Each CBP will be based on consideration of: (i) an epidemiological cut-off value (ECOFF) – the highest MIC that defines the upper end of the wild-type MIC distribution; (ii) a PK/PD breakpoint obtained from pre-clinical pharmacokinetic data [this PK/PD break-point is the highest possible MIC for which a given percentage of animals in the target population achieves a critical value for the selected PK/PD index (fAUC/MIC or fT > MIC)] and (iii) when possible, a clinical cut-off, that is the relationship between MIC and clinical cure. For the latter, VetCAST acknowledges the paucity of such data in veterinary medicine. When a CBP cannot be established, VetCAST will recommend use of ECOFF as surrogate. For decision steps, VetCAST will follow EUCAST procedures involving transparency, consensus and independence. VetCAST will ensure freely available dissemination of information, concerning standards, guidelines, ECOFF, PK/PD breakpoints, CBPs and other relevant information for AST implementation. Finally, after establishing a CBP, VetCAST will promulgate expert comments and/or recommendations associated with CBPs to facilitate their sound implementation in a clinical setting.

Interaction of Antibiotics with Innate Host Defense Factors against Salmonella enterica Serotype Newport.

This study examines the pharmacodynamics of antimicrobials that are used to treat Salmonella with each other and with key components of the innate immune system. Antimicrobial synergy was assessed using time-kill and checkerboard assays. Antimicrobial interactions with innate immunity were studied by employing cathelicidin LL-37, whole-blood, and neutrophil killing assays. Ceftriaxone and ciprofloxacin were found to be synergistic in vitro against Salmonella enterica serotype Newport. Ceftriaxone, ciprofloxacin, and azithromycin each demonstrated synergy with the human cathelicidin defense peptide LL-37 in killing Salmonella. Exposure of Salmonella to sub-MICs of ceftriaxone resulted in enhanced susceptibility to LL-37, whole blood, and neutrophil killing. The activity of antibiotics in vivo against Salmonella may be underestimated in bacteriologic media lacking components of innate immunity. The pharmacodynamic interactions of antibiotics used to treat Salmonella with each other and with components of innate immunity warrant further study in light of recent findings showing in vivo selection of antimicrobial resistance by single agents in this pathogen. IMPORTANCE It is becoming increasingly understood that the current paradigms of in vitro antimicrobial susceptibility testing may have significant shortcomings in predicting activity in vivo. This study evaluated the activity of several antibiotics alone and in combination against clinical isolates of Salmonella enterica serotype Newport (meningitis case) utilizing both conventional and physiological media. In addition, the interactions of these antibiotics with components of the innate immune system were evaluated. Azithromycin, which has performed quite well clinically despite high MICs in conventional media, was shown to be more active in physiological media and to enhance innate immune system killing. Alternatively, chloramphenicol did not show enhanced immune system killing, paralleling its inferior clinical performance to other antibiotics that have been used to treat Salmonella meningitis. These findings are important additions to the building understanding of current in vitro antimicrobial assay limitations that hopefully will amount to future improvements in these assays to better predict clinical efficacy and activity in vivo.