Reliability of E-Tests and the Phoenix Automated Method in Assessing Susceptibility to IV Fosfomycin-Comparative Studies Relative to the Reference Method.

The agar dilution method (ADM) recommended for IV fosfomycin (IV FOS) is complex and labor-intensive. Keeping in mind the reality of everyday laboratory work, we have evaluated the agreement of IV FOS susceptibility results obtained using the E-test and the Phoenix system with the results obtained using the ADM. MATERIALS AND METHODS: The tests were performed on 860 strains. To evaluate susceptibility to IV FOS, BioMerieux E-tests (bioMerieux, Warsaw, Poland), BD Phoenix panels (BD Phoenix, Sparks, MD, USA), and the ADM were used. Clinical interpretation was performed in accordance with EUCAST Guidance (v12.0, 2021). The significance of the E-test and the Phoenix was analyzed in relation to the ADM by defining categorical agreement (CA), major error (ME), and very major error (VME). Essential agreement (EA) has also been defined for the E-test. A method was considered reliable, in accordance with ISO 20776-2:2007, when CA and EA were above 89.9% and VME was <3%. RESULTS: A categorical agreement of >98.9% was demonstrated between the E-test and the ADM for overall strains and for Echerichia coli, ESBL-producing Enterobacterales, and Staphylococcus aureus, while between the Phoenix and the ADM, a CA of >98.9% was shown only for Escherichia coli, Staphylococcus aureus, and Proteus spp. A very major error rate of <3% was obtained only for Staphylococcus aureus and MBL-producing Pseudomonas evaluated by both the E-test and the Phoenix. An essential agreement of >98.9% between the E-test and the ADM has not been demonstrated for any of the tested groups of strains. The Phoenix yielded more VMEs than the E-test (50 and 46, respectively). The highest VME rate was demonstrated using the Phoenix method for Enterobacter spp. (53.83%). CONCLUSIONS: Both the E-test and the Phoenix have turned out to be reliable in assessing IV FOS susceptibility only for Staphylococcus aureus (CA > 89.9% and VME < 3%). For the remaining tested groups of strains and genera, the simultaneous high CA rate and low VME rate required by ISO were not achieved. Both methods fared particularly badly in detecting strains resistant to IV.

Assessment of the Susceptibility of Clinical Gram-Negative and Gram-Positive Bacterial Strains to Fosfomycin and Significance of This Antibiotic in Infection Treatment.

Multidrug resistance of bacteria has prompted intensive development work on new medicines, but also the search for effective options among the oldest antibiotics. Although intravenous fosfomycin (IVFOS) seems to be an interesting proposal, the recommended agar dilution method for susceptibility determination poses a major problem in routine diagnostic testing. As a consequence, there is a lack of comprehensive data on the frequency of isolation of susceptible or resistant strains. This fact triggered the disposition of EUCAST concerning the revision of IVFOS breakpoints (BPs), including withdrawal of BPs for Enterobacterales (excluding E. coli) and coagulase-negative staphylococci. Therefore, the aim of this study was to assess the activity of fosfomycin against numerous clinical strains using recommended methods. Materials and methods: A total of 997 bacterial strains were tested from the following genera: Enterobacterales, Pseudomonas spp., Staphylococcus spp., Acinetobacter spp., and Enterococcus spp., for which there are currently no BPs. The strains were isolated from various clinical materials from patients hospitalized in five hospitals. During the investigation, the recommended agar dilution method was used. Susceptibility to other antibiotics and resistance mechanisms were determined using an automatic method (Phoenix) the disk diffusion method, and E-tests. MIC values of fosfomycin were estimated for all strains and for susceptible and multidrug-resistant (MDR) strains individually. Results: Except for Acinetobacter and Enterococcus, 83% of the strains were susceptible to IVFOS, including the largest percentage of S. aureus and E. coli. Klebsiella spp. turned out to be the least susceptible strains (66%). The highest proportion of susceptibility to fosfomycin was found among strains that were sensitive to other antibiotics (80.9%), and the lowest was found among Gram-negative carbapenemase-producing bacteria (55.6%) and ESBL+ bacteria (61.6%). The MIC evaluation revealed the lowest MIC50 and MIC90 values for S. aureus (0.5 mg/L and 1 mg/L, respectively) and E. coli (4 mg/L and 32 mg/L, respectively). The highest values of MIC50 were found for Acinetobacter spp. (256 mg/L), while the highest values of MIC90 were found for Acinetobacter spp. and Klebsiella spp. (256 mg/L and 512 mg/L, respectively). Conclusions: IVFOS appears to be suitable for the treatment of many infections, including the empirical treatment of polymicrobial infections and those caused by MDR strains, since the sensitivity of the studied strains to this antibiotic in different groups ranged from 66% to as much as 99%. Sensitivity to fosfomycin was also demonstrated by 60% of carbapenem-resistant strains; therefore, IVFOS is one of the few therapeutic options that can be effective against the most resistant Gram-negative rods. In light of the general consultation posted by EUCAST, obtaining data such as IVFOS MIC value distributions may be vital for the decision of implementing fosfomycin into breakpoint tables.

Activity of Liquid and Volatile Fractions of Essential Oils against Biofilm Formed by Selected Reference Strains on Polystyrene and Hydroxyapatite Surfaces.

Biofilms are surface-attached, structured microbial communities displaying higher tolerance to antimicrobial agents in comparison to planktonic cells. An estimated 80% of all infections are thought to be biofilm-related. The drying pipeline of new antibiotics efficient against biofilm-forming pathogens urges the search for alternative routes of treatment. Essential Oils (EOs), extracted from medicinally important plants, are a reservoir of bioactive compounds that may serve as a foothold in investigating novel antibiofilm compounds. The aim of this study was to compare antimicrobial activity of liquid and volatile fractions of tested EOs against biofilm-forming pathogens using different techniques. In this research, we tested five EOs, extracted from Syzygium aromaticum L., Boswelia serrata Roxb., Juniperus virginiana L., Pelargonium graveolens L. and Melaleuca alternifolia Cheel., against planktonic and biofilm forms of five selected reference strains, namely Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans. To obtain cohesive results, we applied four various methodological approaches: to assess the activity of the liquid fraction of EOs, disc diffusion and the microdilution method were applied; to test EOs' volatile fraction, the AntiBioVol assay and modified Antibiofilm Dressing Activity Measurement (A.D.A.M.) were used. The molecular composition and dynamics of antimicrobial substances released from specific EOs was measured using Gas Chromatography-Mass Spectrometry (GC-MS). The antimicrobial potency of EO's volatile fraction against biofilm formed by tested strains differed from that of the liquid fraction and was related to the molecular weight of volatile compounds. The liquid fraction of CW-EO and volatile fraction of F-EO acted in the strongest manner against biofilm of C. albicans. The addition of 0.5% Tween 20 to liquid phase, enhanced activity of G-EO against E. coli and K. pneumoniae biofilm. EO activity depended on the microbial species it was applied against and the chosen assessment methodology. While all tested EOs have shown a certain level of antimicrobial and antibiofilm effect, our results indicate that the choice of EO to be applied against a specific biofilm-forming pathogen requires careful consideration with regard to the above-listed aspects. Nevertheless, the results presented in this research contribute to the growing body of evidence indicating the beneficial effects of EOs, which may be applied to fight biofilm-forming pathogens.

The Minimum Inhibitory Concentration of Antibiotics: Methods, Interpretation, Clinical Relevance.

Inefficiency of medical therapies used in order to cure patients with bacterial infections requires not only to actively look for new therapeutic strategies but also to carefully select antibiotics based on variety of parameters, including microbiological. Minimal inhibitory concentration (MIC) defines in vitro levels of susceptibility or resistance of specific bacterial strains to applied antibiotic. Reliable assessment of MIC has a significant impact on the choice of a therapeutic strategy, which affects efficiency of an infection therapy. In order to obtain credible MIC, many elements must be considered, such as proper method choice, adherence to labeling rules, and competent interpretation of the results. In this paper, two methods have been discussed: dilution and gradient used for MIC estimation. Factors which affect MIC results along with the interpretation guidelines have been described. Furthermore, opportunities to utilize MIC in clinical practice, with pharmacokinetic /pharmacodynamic parameters taken into consideration, have been investigated. Due to problems related to PK determination in individual patients, statistical estimation of the possibility of achievement of the PK/PD index, based on the Monte Carlo, was discussed. In order to provide comprehensive insights, the possible limitations of MIC, which scientists are aware of, have been outlined.

In Vitro Evaluation of Polihexanide, Octenidine and NaClO/HClO-Based Antiseptics against Biofilm Formed by Wound Pathogens.

Chronic wounds complicated with biofilm formed by pathogens remain one of the most significant challenges of contemporary medicine. The application of topical antiseptic solutions against wound biofilm has been gaining increasing interest among clinical practitioners and scientific researchers. This paper compares the activity of polyhexanide-, octenidine- and hypochlorite/hypochlorous acid-based antiseptics against biofilm formed by clinical strains of Candida albicans, Staphylococcus aureus and Pseudomonas aeruginosa. The analyses included both standard techniques utilizing polystyrene plates and self-designed biocellulose-based models in which a biofilm formed by pathogens was formed on an elastic, fibrinous surface covered with a fibroblast layer. The obtained results show high antibiofilm activity of polihexanide- and octenidine-based antiseptics and lack or weak antibiofilm activity of hypochlorite-based antiseptic of total chlorine content equal to 80 parts per million. The data presented in this paper indicate that polihexanide- or octenidine-based antiseptics are highly useful in the treatment of biofilm, while hypochlorite-based antiseptics with low chlorine content may be applied for wound rinsing but not when specific antibiofilm activity is required.

Frequency of isolation and drug susceptibility of bacterial strains isolated from child oncohematological patients 2011-2014: A single center study.

BACKGROUND: Infections in pediatric patients with oncohematological diseases pose a huge therapeutic and diagnostic problem. OBJECTIVES: The aim of the study was to investigate the etiology of bacteremia and the antibiotic susceptibility of pathogenic and colonizing bacterial strains in pediatric oncohematological patients. MATERIAL AND METHODS: In the period 2011-2014, 17,209 positive test results, including 1,129 positive blood cultures, were subjected to a detailed analysis. The assessment of drug susceptibility was conducted in accordance with the CLSI (American), EUCAST (European), and KORLD (Polish) recommendations. RESULTS: A high percentage (86-91%) of negative blood culture results was demonstrated. A predominance of Gram-positive bacteria was seen in all years (60-70%) in contrast to Gram-negative strains (30-40%). Coagulase-negative staphylococci (CNS) were the strains most frequently isolated from blood (41-47%) among all bacterial strains. Susceptibility to linezolid and vancomycin was 96-100%, and to teicoplanin 82-96%. Methicyllin-resistant coagulase-negative Staphylococcus (MRCNS) were isolated in 77-86%. All Staphylococcus aureus (S. aureus) strains were susceptible to glycopeptides and linezolid, while Enterococcus spp. was susceptible to linezolid. Apart from the year 2014, no methicillin-resistant S. aureus (MRSA) were isolated. Enterobacteriaceae (EN) were the most susceptible to imipenem and meropenem (91-100%) as well as to amikacin (77-93%). From 2013 to 2014, non-fermentative rods (NF) isolated from blood were less susceptible to imipenem and meropenem (71% and 67-71%, respectively) than to other antibiotics. It has been shown that strains isolated from blood have a statistically significantly different susceptibility to antibiotics (CNS and EN are less and NF is more susceptible) than those existing as colonizing flora. CONCLUSIONS: Our results show that choosing appropriate antibiotics for treating infection in children with oncohematological diseases based on antibiograms for colonizing flora may be difficult because they may not take into account the more resistant strains. According to the antibiotic susceptibility of the strains isolated from blood in our center, the most viable active empirical and carbapenem-saving therapy could be conducted with piperacillin/tazobactam or cefepime.

Therapeutic drug monitoring of amikacin in septic patients.

INTRODUCTION: Use of higher than standard doses of amikacin (AMK) has been proposed during sepsis, especially to treat less susceptible bacterial strains. However, few data are available on drug concentrations during prolonged therapy and on potential adverse events related to this strategy. METHODS: Sixty-three critically ill patients who required AMK administration for the treatment of severe infection were included in this study. After a loading dose (LD, 18 to 30 mg/kg), the daily regimen was adapted using therapeutic drug monitoring (TDM) of both peak (Cpeak) and trough (Cmin) concentrations. Target concentrations had to give a ratio of at least 8 between Cpeak and the minimal inhibitory concentration (MIC) of the isolated pathogen. A Cmin >5 mg/L was considered as potentially nephrotoxic. We recorded clinical and microbiological responses, the development of acute kidney injury (AKI) during therapy and ICU mortality. RESULTS: The median AMK LD was 1500 (750 to 2400) mg, which resulted in a Cpeak/MIC ≥8 in 40 (63%) patients. Increasing the dose in the 23 patients with a Cpeak/MIC <8 resulted in optimal Cpeak/MIC in 15 of these patients (79%). In 23 patients (37%), Cmin was >5 mg/L after the LD, notably in the presence of altered renal function at the onset of therapy, needing prolongation of drug administration. Overall, only 11 patients (17%) required no dose or interval adjustment during AMK therapy. Clinical cure (32/37 (86%) vs. 16/23 (70%), P = 0.18)) and microbiological eradication (29/35 (83%) vs. 14/23 (61%), P = 0.07) were higher in patients with an initial optimal Cpeak/MIC than in the other patients. The proportion of patients with clinical cure significantly improved as the Cpeak/MIC increased (P = 0.006). Also, increased time to optimal Cpeak was associated with worse microbiological and clinical results. AKI was identified in 15 patients (24%) during AMK therapy; 12 of these patients already had altered renal function before drug administration. Survivors (n = 47) had similar initial Cpeak/MIC ratios but lower Cmin values compared to nonsurvivors. CONCLUSIONS: TDM resulted in adjustment of AMK therapy in most of our septic patients. Early achievement of an optimal Cpeak/MIC ratio may have an impact on clinical and microbiological responses, but not on outcome. In patients with impaired renal function prior to treatment, AMK therapy may be associated with a further decline in renal function.

Enterococcus in wound infections: virulence and antimicrobial resistance.

Enterococci, a complex group of facultative pathogens have become increasingly isolated in various hospital settings. They are considerable frequently cultured from traumatic and surgical wounds. We investigated 57 strains of the species E. faecalis, E. faecium and E. casseliflavus isolated from infected wounds. Their ability to produce virulence factors and their sensitivity to antibiotics were evaluated using phenotypic and genotyping methods. In the phenotype studies, significant portion of the isolates produced biofilm (66.7%) and gelatinase (36.8%). Nearly 30% of the strains expressed hemolytic properties. Only a few produced DNAse (15.8%) and lipase (7.0%). The genes esp, gelE, cylA, cylB, cylM and agg were detected in most of the isolates (38.6-87.7%). All the isolated enterococci were susceptible to vancomycin and were characterized by their low resistance to antibiotics, except aminoglycosides (HLR).

Continuous infusion of piperacillin/tazobactam in ventilator-associated pneumonia: a pilot study on efficacy and costs.

Ventilator-associated pneumonia (VAP) occurs in nearly one-third of mechanically ventilated patients in the Intensive Care Unit. Piperacillin/tazobactam (TZP) is currently recommended in the empirical treatment of VAP, but intermittent dosing may result in inadequate serum concentrations. The efficacy and costs of continuous infusion (CI) of TZP, using therapeutic drug monitoring for real-time dose adjustment, was assessed in a prospective pilot study of 16 patients with VAP. TZP was given as a loading dose of 2.0/0.25 g followed by a CI of 10.0/1.25g daily. Rapid antimicrobial susceptibility testing was used to determine the minimum inhibitory concentration (MIC) of the pathogens. TZP concentrations were determined by high-pressure liquid chromatography before and at 1, 6, 12, 24, 48, 72 and 96 h after the onset of administration. Dosages were adjusted to maintain piperacillin concentrations four-fold above the MIC (T>4 × MIC) of the pathogen, with a maximum dose of 16.0/2.0 g. The cost of the total TZP administered was compared with the cost of a standard TZP regimen (16.0/2.0 g) if given over the same period of time. The median MIC for TZP was 1 µg/mL (range 0.025-32 µg/mL). TZP concentrations were adequate for 71% of pathogens on the first day of therapy. Clinical cure was achieved in 9/10 patients who had adequate drug concentrations and in 3/6 patients with insufficient levels. The daily dose of TZP received by CI was 37.5% less than that of a standard regimen, which corresponds to a saving of €15 on daily therapy costs compared with the standard regimen. In conclusion, CI of TZP achieved optimal drug concentrations in most patients with VAP, with a favourable impact on costs. Adequate drug concentrations were achieved for MIC ≤ 4 µg/mL, but higher dosages should be considered for the treatment of pathogens with low susceptibility thresholds.

[Estimation of level of aminoglycosides susceptibility of bacterial strains isolated from patients with systemic and general infections]. / Ocena stopnia wrazliwosci na aminoglikozydy szczepów bakteryjnych izolowanych od chorych z zakazeniami ukladowymi i uogólnionymi.

Susceptibility tests to aminoglycosides of 200 bacterial strains isolated in period 1.01.2007-30.04.2008 from hospitalized patients in different Wroclaw clinics were performed. Among tested strains were 96 Gram-negative rods belonging to the Enterobacteriaceae family, 74 non-fermentative rods and 30 strains of Staphylococcus aureus. The results of antibiotic susceptibility determined by serial antibiotic dilution in solid medium indicated that among Enterobacteriaceae the most susceptible to aminoglycosides were E. coli (90-95%) and least susceptible Enterobacter spp., Citrobacter spp. and Serratia spp (62-76%). Among non-fermentative rods, Pseudomonas aeruginosa was more susceptible than Acinetobacter spp. 97% strains of Staphylococcus aureus were susceptible to all aminoglycosides. Enterobacteriaceae strains isolated from adults were more resistant to aminoglycosides than isolated from children and non-fermentative rods isolated from adults more susceptible to netilmicin. Among S. aureus only one strains was MRSA and aminoglycoside resistant. Twenty two of 96 examined strains belonging to Enterobacteriaceae and 46 of 74 examined non-fermentative rods were resistant at least to one aminoglycoside. Predominant resistance patterns were as follow: simultaneous resistance to all aminoglycosides and resistance to gentamicin and netilmicin with susceptible to amikacin. High level resistance (MIC > or = 256 microg/ml) was found in 23 gentamicin resistant strains (12 rods of Enterobacteriaceae and 11 non-fermentative rods), in 33 netilmicin resistant strains (respectively 10 and 21 strains) and in 14 amikacin resistant Gram-negative rods (respectively 9 and 5 strains). Among 96 rods of Enterobacteriaceae family 19 ESBL (+) strains were found. High level of resistance (MIC > or = 256 microg/ml) was found in 10 ESBL (+) strains.

[Conjugative transfer frequency of resistance genes from ESBL-producing Enterobacteriaceae strains isolated from patients hospitalized in pediatric wards]. / Czestosc koniugacyjnego transferu genów lekoopornosci z ESBL-dodatnich paleczek Enterobacteriaceae izolowanych od chorych z oddzialow pediatrycznych.

The aim of this study was to evaluate the transfer frequency of plasmids encoding extended-spectrum beta-lactamases (ESBLs) from clinical isolates of Enterobacteriaceae to E. coli K12 C600 recipient strain. Additionally, resistance patterns to antimicrobial drugs of the isolates as well as transconjugants were analyzed. Fifty-four clinical strains belonging to the Enterobacteriaceae family were isolated from children hospitalized in Medical University Hospital in Wroclaw. All the strains studied were identified in automatic ATB system using ID32E tests. Besides, they were ESBL-positive as was confirmed by the double-disc synergy test (DDST). The minimal inhibitory concentration (MIC) was determined for twelve selected antibiotics and chemotherapeutics. The majority of the strains (87%) were able to transfer plasmid-mediated ESBL to E. coli K12 C600 recipient strain with a frequencies ranged from 10(-5) to 10(-1) per donor cell. All the isolates studied as well as their transconjugants were susceptible to imipenem, meropenem and norfloxacin (MIC <1mg/L). On the other hand, these strains displayed high level of resistance (MIC 512 - >1024 mg/L) to cefotaxime, ceftriaxone, gentamycin, amikacin and cotrimoxazole. Genetic markers conferring resistance to aminoglycosides and cotrimoxazole were often co-transferred to recipient strain in conjugation process.

[Susceptibility to antimicrobial agents of enteroaggregative Escherichia coli strains isolated from children with diarrhea]. / Wrazliwosc na antybiotyki i chemioterapeutyki enteroagregacyjnych szczepów Escherichia coli (EAEC) izolowanych od dzieci z biegunka.

Enteroaggregative Escherichia coli (EAEC) are responsible for infant's persistent secretory diarrhea, traveler's diarrhea and outbreaks. Since isolated all over the world EAEC strains show a low to high level of resistance to antimicrobial agents, the aim of the study was to determine antimicrobial susceptibility and beta-lactamases extended spectrum (ESBL) production in vitro among 55 enteroaggregative E. coli strains isolated from children with diarrhea. Among the 17 antimicrobial agents tested, most of them showed good in vitro activity against EAEC isolates examined, apart from ampicylin and piperacylin (about 30% resistant EAEC strains). Three of 55 examined EAEC isolates were ESBL--producing strains. Because of the presence of ESBL--producing as well as resistant to antimicrobial agents EAEC isolates among enteroaggregative E. coli strains examined, treatment with antibiotics should be undertaken according to antibiogram.