Disk Susceptibility Testing Research Articles

Antimicrobial Activity of Diverse Chemotypes of Lippia graveolens Against Aeromonas hydrophila Isolated from Oreochromis niloticus

[Objective] This study aimed to evaluate the antimicrobial efficacy of essential oil (EO) from diverse chemotypes of Lippia graveolens against oxytetracycline-resistant Aeromonas hydrophila, which primarily affects the tilapia aquaculture (O. niloticus) in Guatemala. [Methodology] L. graveolens were collected in three departments in Guatemala. The EO was obtained by hydrodistillation and characterized using gas chromatography and mass spectrometry (GC/MS). Subsequently, an antimicrobial assay was conducted using disk and dilution susceptibility tests and evaluation of synergistic interactions among the chemotypes. Each test was performed in triplicate. [Results] The analysis revealed the presence of twenty-seven compounds in the EO obtained from the chemotypes, with the main class being monoterpenes. The major constituents identified were cis-Dihydro-β-terpineol (8.84%) in chemotype I, carvacrol (51.82%) in chemotype II, and thymol (79.62%) in chemotype III. All EO chemotypes of L. graveolens demonstrated the ability to inhibit the A. hydrophila growth. Thymol chemotype exhibited the strongest inhibitory effect against bacterial growth, with a minimum inhibitory concentration (MIC) of 92.4 µg/mL and a minimum bactericidal concentration (MBC) of 184.8 µg/mL. Furthermore, the results suggest that there is no synergistic or additive effect when combining different chemotypes of L. graveolens. [Conclusions] This is the first report of L. graveolens chemotypes exhibiting antimicrobial activity against oxytetracycline-resistant A. hydrophila. The findings suggest that the chemotype thymol could be a potential treatment for infections in the tilapia aquaculture in Guatemala.

Evaluation of antimicrobial susceptibility tests for Acinetobacter and Pseudomonas species using disks containing a high dose of meropenem

The emergence and dissemination of carbapenem-resistant species of Acinetobacter and Pseudomonas have become a serious health concern. Routine antimicrobial disk susceptibility tests in clinical laboratories cannot distinguish between isolates that are highly carbapenem-resistant and those that are moderately carbapenem-resistant. The present study describes antimicrobial susceptibility tests using disks containing high doses (1000 μg) of meropenem. The diameters of inhibition zones were significantly negatively correlated with the MICs of Pseudomonas and Acinetobacter species for meropenem (R2: 0.93 and 0.91, respectively) and imipenem (R2: 0.75 and 0.84, respectively). Double disk synergy tests using clavulanic acid or sodium mercaptoacetate can detect ESBL or MBL producers. Susceptibility tests using disks containing high doses of meropenem can easily detect highly carbapenem-resistant isolates in a quantitative manner. These disks may be useful in bacteriological laboratories because of their technical ease, stability, and relatively low cost.

In Vitro Elution of Gentamicin from CERAMENT® G Has an Antimicrobial Effect on Bacteria With Various Levels of Gentamicin Resistance Found in Fracture-related Infection.

Fracture-related infection is a serious complication after trauma. CERAMENT® G combines dead-space management with local release of gentamicin in a single-stage procedure. Bacterial resistance against antibiotics is increasing. The local effect of CERAMENT® G on bacteria resistant to systemically administered gentamicin is unknown. (1) What is the in vitro elution pattern of gentamicin from CERAMENT® G using a full washout model? (2) What is the in vitro antimicrobial activity (zone of inhibition) of CERAMENT® G against bacterial isolates found in fracture-related infection with different susceptibility levels toward gentamicin? Elution of gentamicin from CERAMENT® G was determined in vitro over a period of 2 months. Elution experiments were performed in fivefold, with gentamicin being sampled in threefold at 19 different timepoints within 2 months. Antimicrobial activity was determined using the four most-frequently cultured bacterial species found in fracture-related infection: Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Enterobacter cloacae . For each of the species, four different isolates with a different susceptibility to gentamicin were used. According to the European Committee on Antimicrobial Susceptibility Testing, the susceptibility of each isolate was classified into four different groups: fully susceptible (minimum inhibitory concentration 0.064 to 4 mg/L), minimally resistant (minimum inhibitory concentration 4 to 16 mg/L), moderately resistant (minimum inhibitory concentration 8 to 96 mg/L), and highly resistant (minimum inhibitory concentration 24 to 1024 mg/L), depending on each organism. The antimicrobial activity of CERAMENT® G was determined according to the European Committee on Antimicrobial Susceptibility Testing disk protocol. The experiment was performed in fivefold for each isolate. The zone of inhibition was compared between each bacterial isolate and within each of the four separate species. Nonlinear regression statistics were calculated between the zone of interest and logarithmic minimum inhibitory concentration for each bacterial species. After 24 hours, 95% of all available gentamicin was eluted, and gentamicin was still detectable after 2 months. CERAMENT® G showed antimicrobial activity against all bacterial species; only S taphylococcus aureus (with a minimum inhibitory concentration > 1024 mg/L) was not susceptible. The zone of interest of the different bacterial isolates was correlated with the logarithmic minimum inhibitory concentration. CERAMENT® G offers a bone substitute capable of releasing high levels of gentamicin within a short period of time. This study shows that CERAMENT® G has antimicrobial activity against bacterial isolates that are resistant to gentamicin when systemically administered. This finding raises the question of whether European Committee on Antimicrobial Susceptibility Testing cutoff points for systemic application are useful for the use of local CERAMENT® G. Standardized experiments to determine local antibiotic antimicrobial activity in fracture-related infection treatment are needed to form guidelines for the use of local antibiotics and ultimately improve fracture-related infection treatment. Local concentrations of gentamicin with CERAMENT® G are much higher than when systemically administered. It seems effective against certain bacterial strains that are not affected by systemically reachable concentrations of gentamicin. CERAMENT® G might still be effective when bacteria that are resistant to systemically administered concentrations of gentamicin are occulated from patients with fracture-related infection.

Correlation between oxacillin MIC values with those of linezolid against clinical isolates of MRSA

Infection by methicillin-resistant (MRSA) is a great threat to medical care facilities and also in communities, due to its multidrug resistance to commonly used antimicrobial agents. The overuse of glycopeptide antibiotics, such as vancomycin and linezolid, has led to the emergence of reduced susceptibility to these anti-MRSA agents, which in turn may lead to therapeutic failure. This study has been conducted to explore the correlation if any, of MIC of oxacillin to the MIC of linezolid in clinically significant isolates of MRSA in a tertiary care hospital.: The study was carried out over the period of two months after obtaining a waiver of consent from the Institute Ethics Committee. Seventy-five clinically significant MRSA isolates were included in the study. All MRSA isolates were subjected to cefoxitin and linezolid antibiotic disk susceptibility testing. Minimum inhibitory concentration (MIC) to oxacillin and linezolid was performed by the agar dilution method. The MIC and MIC were also recorded both for oxacillin and linezolid MIC among these MRSA isolates. Correlation between oxacillin MIC and linezolid MIC was estimated using the Pearson correlation coefficient, r.Themajority of MRSA isolates (41%) were isolated from skin and soft tissue infections (SSTIs) (40%). MIC values for oxacillin ranged from 4 μg/mL to >32μg/mL and MIC values for linezolid ranged from ≤ 0.25µg/mL to 4 µg/mL. The majority of these isolates (40%) had linezolid MIC of ≤ 0.25µg/mL. All the isolates were uniformly susceptible to linezolid. Pearson correlation coefficient, r was 0.41, between oxacillin MICs and linezolid MICs, which indicated poor correlation. Although we did not observe any resistance to linezolid among the MRSA isolates, we should monitor carefully the antibiotic selection pressure and creeping MIC, to aid in the early detection of the emergence of resistance.

Highly stable and fast-dissolving ascorbic acid-loaded microneedles.

Ascorbic acid has many benefits to the skin. Numerous attempts to promote its topical delivery show great challenges since its chemical instability and poor skin impermeability. Microneedle delivery is a simple, safe, painless and effective means to deliver therapeutic or nourishing molecules into the skin. The purpose of this study was twofold: (a) to develop a new formulation of ascorbic acid-loaded microneedles to enhance ascorbic acid stability by investigating an optimal amount of polyethyleneimine as an additive to the dextran-based microneedle formulation and (b) to assess microneedle properties in terms of dissolving rate, skin penetration ability, biocompatibility and antimicrobial activity. The microneedles formulated with ascorbic acid and varied polyethyleneimine concentrations were fabricated and subsequently tested for ascorbic acid stability using 2,2-diphenyl-1-picrylhydrazyl assay. The dissolution rate and skin penetration depth were investigated in porcine skin and the reconstructed human full-thickness skin model respectively. The skin irritation tests were done according to the Organisation for Economic Co-operation and Development Test Guideline No. 439. An antimicrobial disc susceptibility test was performed against Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis. Among varied amounts of 0%, 1.5%, 3.0% and 4.5% (w/v), the 3.0% polyethyleneimine showed the most desirable characteristics, including well-preserved shape integrity after demoulding, significantly improved stability of ascorbic acid (p < 0.001) from 33% to 96% antioxidant activity after 8 weeks of storage at 40°C, increased dissolving rate (p < 0.001) by being completely dissolved within 2 min after the skin insertion, passing skin penetration and biocompatibility tests as well as having a broad spectrum of antimicrobial property. With a safety profile and enhanced properties, the new formulation of ascorbic acid-loaded microneedles shows outstanding potential as commercially available cosmetics and healthcare products.

Cutibacterium Acnes (Formerly Propionibacterium Acnes ): Friend or Foe?

Cutibacterium acnes protects skin homeostasis. The species has three subspecies, and associations between C. acnes subsp. acnes and acne, C. acnes subsp. defendens and prostate cancer, and C. acnes subsp. elongatum and progressive macular hypomelanosis have recently been suggested. Different phylotypes/clonal complexes may cause prosthetic joint and other infections, and virulence factors such as fimbriae, biofilms, multidrug-resistance plasmids, porphyrin, Christie-Atkins-Munch-Petersen factors and cytotoxicity contribute to infections. Isolates are subtyped by multiplex PCR or multi- or single-locus sequence typing; however, these methods could be better synchronized. Resistance of acneic strains to macrolides (25.0-73.0%), clindamycin (10.0-59.0%) and tetracyclines (up to 37.0%) is worrisome, but susceptibility testing is now facilitated by European Committee on Antimicrobial Susceptibility Testing disk diffusion breakpoints. New therapeutic approaches include sarecycline, antimicrobial peptides and bacteriophages.

Antibiotic resistance pattern of Methicillin-resistant Staphylococcus aureus in asymptomatic prisoners of Kathmandu valley

bjectives: The purpose of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus and its antimicrobial resistance among the prisoners within the prisons of Kathmandu valley. Methods: A total of 320 nasal samples were collected from the prisoners. S. aureus was confirmed by using standard microbiological techniques including culture and biochemical tests. MRSA was confirmed using cefoxitin disc and antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion method results were interpreted according to the guidelines of Clinical Laboratory Standard Institute (CLSI). Results: Out of 320 samples, 41 (12.82%) isolates were confirmed S. aureus and 8 (19.51%) of them were screened as MRSA positive. Highest percentage of MRSA was found in the samples collected from Nakhu prison 6 (75%) followed by Central prison 1 (12.5%) and Juvenile Correctional Home 1 (12.5%). Majority of the of MRSA isolates (62.5%) were also found to be multiple drug-resistant strains. All of the MRSA (100%) were sensitive towards linezolid and resistant towards ciprofloxacin. Conclusion: The presence of methicillin-resistant S. aureus among the prisoners indicates the emergence of drug resistance in a vulnerable population which may lead to hazardous disease breakouts in the prison and even in the community after their release. Regular monitoring and its minimization should be done in order to control its dissemination.

Environmental surveillance of antimicrobial resistance in a rapidly developing catchment.

Development and spread of AMR from various sources such as hospitals, pharmaceutical industries, animal farms and human habitations is not well understood. We conducted a pilot study to assess the prevalence of AMR by taking a case of rapidly developing catchment in Western India. For this, we selected four sub-catchments/regions with a dominant source of antibiotics, viz. areas with (a) dense poultry farms (4 farms/km2), (b) sparse number of poultry farms (2 farms/km2), (c) agricultural fields and (d) habitation (village). The environmental samples (soil, litter and water) were subjected to Kirby-Bauer/antibiotic disc susceptibility test to assess the resistance pattern in the bacterial species. Preliminary investigations showed the presence of seven multidrug-resistant bacterial species in the litter from poultry farms, with five species having a MAR index greater than 0.2. No evidence of AMR was observed in the vicinity (water and soil) of the poultry farms. This could be attributed to the rigorous disinfection protocols followed at the poultry farms to prevent infection in the fresh batch of chickens. However, in agricultural fields where the litter is used as manure, seven multiple drugs resistant with two species scoring a MAR index greater than 0.2 were observed. MAR index of less than 0.2 was observed for Escherichia coli and Enterobacter species isolated from village and control site (soil), respectively, indicating negligible contamination by antibiotics at sub-catchments. This study provides an approach to investigate the effects of multiple factors on the prevalence of AMR at the catchment scale.

Characterization of Resistance and Virulence of Pasteurella multocida Isolated from Pet Cats in South China

Pasteurella multocida, one of the major zoonotic pathogens, may cause localized cellulitis, bacteremia, septic shock, and other symptoms in people. Accidental bites or scratches in close contact between humans and cats are one of the causes of P. multocida infection in humans. The prevalence of diseases due to P. multocida in humans may be rising as a result of the popularity of cat cafes in China. However, the epidemiology of P. multocida in cat-themed cafes in South China is yet to be reported. P. multocida in 92 healthy cats from four cafes with pet-cat themes was examined in their tonsils and oral cavities. The antibiotic susceptibility of each isolate was evaluated by using the disk-susceptibility testing method. The 19 P. multocida were selected for whole-genome sequencing (WGS), together with the genome data of eight previously described clinical strains isolated from humans, and the analysis of antimicrobial resistance (AMR) determinants, capsular genotyping, MLST genotyping, and virulence gene profiles was carried out. The results showed that 48.91% of cats (45/90) were positive for P. multocida. All isolated P. multocida stains were highly resistant to erythromycin (15 μg) (100%) and nonsensitive to fluoroquinolones (5 μg) (37.38%), while they were all susceptible to penicillin (10 untis), tetracycline (30 μg), doxycycline (30 μg), and chloramphenicol (30 μg). According to WGS analysis, those with the gyrA resistance gene were all insensitive to fluoroquinolone antibiotics. Virulence gene profiles showed that the genes pfhA, nanH, and plpB were practically all present in cats compared to humans. To conclude, increased antibiotic resistance, along with virulence factors, complicates the treatment of P. multocida infection. Thus, clinical treatment for P. multocida infection should be performed more cautiously.

Methicillin-resistant Staphylococcus aureus colonization among medicine and health science students, Arba Minch University, Ethiopia

Individuals with Methicillin-Resistant Staphylococcus aureus (MRSA) colonized nasal cavities were at greater risk of developing the infection and can serve as potential reservoirs of transmission. Aim of this study is to determine the extent of nasal carriage and associated factors linked to MRSA in medical and health science students of Arba Minch University (AMU), Ethiopia, who are much prone. An institution based cross-sectional study was conducted at AMU from 01st August through 30th November, 2020 by means of a systematic sampling technique using a structured questionnaire. Nasal swabs samples were collected and S. aureus were identified following standard microbiological methods. Methicillin resistance was tested using cefoxitin disk and antimicrobial susceptibility tests were performed by Kirby-Bauer disk diffusion. Biofilm forming ability was phenotypically detected by micro-titer plate assay. Descriptive statistics and multivariable logistic regression analysis were done by Statistical Package for Social Service (SPSS) version 25. The overall prevalence of Staphylococcus aureus and MRSA were 27.1% (70/258) and 7.4% (19/258) respectively. Methicillin-Resistant S. aureus carriage were higher among medical interns, 16.9% (11/65). Isolates in general were co-resistant to antibiotics, such as trimethoprim-sulfamethoxazole (63.2%) and tetracycline (48.4%). Multidrug resistance (MDR) were observed among 52.6% (10/19) of the isolates. Besides, 31.4% (6/19) of MRSA were biofilm producers and all of them were MDR. Multivariable analysis showed that mean exposure for > 2 years to hospital settings [p = 0.048, AOR: 4.99, 95% CI 1.01–24.66] and the habit of sharing clothing and sports equipment [p = 0.017, AOR: 5.43, 95% CI 1.35–21.83] were statistically significant. The overall prevalence of nasal colonized MRSA among students were comparatively lower than that observed in other studies done in Ethiopia itself. An alarming factor is that, 60% of MDR-MRSA were biofilm producers.

PREVALENCE OF METALLO-?-LACTAMASE RESISTANT GRAM-NEGATIVE BACILLI IN BENIN CITY, NIGERIA

Detection of metallo-?-lactamase (MBL) is crucial in the management and control of difficult to treat infections. This study aims to determine the prevalence of MBL – producing Gram-negative bacilli among imipenem-resistant multi-drug resistant (MDR) clinical isolates. A total of 224 consecutive MDR Gram-negative bacilli consisting of Escherichia coli (78), Klebsiella species (64), Proteus species (40) and Pseudomonas aeruginosa (40) were used for this study. Imipenem disc susceptibility test was performed on all isolates and MBL detection was performed on imipenem-resistant isolates using standard techniques. Of the 224 MDR isolates tested, 37 (16.52%) were imipenem resistant and the prevalence of this resistant isolates was significantly higher in Ps. aeruginosa (35.00%) compared to others (P = 0.005). Among, the imipenem-resistant isolates, the prevalence of MBL production was highest in Ps. aeruginosa (14.29%) followed by Escherichia coli (11.11%), Klebsiella species (10.00%) and Proteus species (0%). However, the difference in prevalence was not statistically significant (P = 0.8807). Conclusively, Ps. aeruginosa was the most prevalent microorganism that was resistant to imipenem. An overall prevalence of 10.81% of MBL producers was observed among imipenem-resistant microbial isolates. Measures to control and prevent the spread of MBL producing and imipenem-resistant microbial isolates are advocated.

Ensuring robustness in the quality of antibiotic susceptibility test discs for four novel antibiotics.

Antibiotic susceptibility test (AST) discs are used as an in-vitro diagnostic tool to select the appropriate antibiotic to treat an infection. Generally, the concentration of the drug loaded on to the AST discs is measured by studying its activity against quality control organisms. This methodology has several limitations-it is time consuming, requires trained manpower, has a wider acceptance criteria of zone of inhibitions-causing ambiguity in judging smaller variations in drug concentration. To overcome these issues, we have developed and validated high-performance liquid chromatographic (HPLC) methods for the determination of strength of AST discs for in-house researched antibiotics, namely Levonadifloxacin/WCK 771, Nafithromycin/WCK 4873, Cefepime-Tazobactam/WCK 4282, and Cefepime-Zidebactam/WCK 5222. The drugs were extracted from the AST discs using an appropriate solvent. The developed methods are simple, accurate, precise, reproducible, rugged, and robust. They are efficient in terms of time, and can be easily conducted in a quality control laboratory during release as well as stability evaluation of AST disc. Application of HPLC methods for the determination of strength of AST discs ensures flawless quality and, consequently, a better selection of drugs to treat bacterial infections in clinics.

Quantitative Measurement of Enteric Bacteria Load From Public Surfaces in Restaurants in Nnewi-Town

Background: Bio-contamination of surfaces of various items and equipment used by the public is the main cause of disease epidemic. Most outbreaks of infection associated with inanimate objects are caused by items that should be sterile but have been contaminated and are generally known as fomites.&#x0D; Objective: This study is to evaluate the presence of enteric bacteria from different public surfaces in restaurants in Nnewi town.&#x0D; Methodology: Selected public surfaces in eight (8) randomly selected restaurants were analyzed for the presence of enteric bacteria via culture and biochemical tests following a standard criterion. The sample sites included; tables, chairs, counters, sinks and walls. Also, Disc susceptibility test were carried out on the isolates using conventional antibiotics.&#x0D; Results: Eighty-eight (88) bacteria isolates were isolated and identified, and S. aureus showed the highest prevlence 29(33%), followed by E. coli 15(17%), Proteus spp 13(15%), Klebsiella spp 12(14%), Pseudomonas spp 10(11%), and Coagulase-negative Staphylococci 9(10%). Tables and sinks were the most contaminated fomites each constituting of 19(22%) of the total bacteria isolated in this study. Escherichia coli, Pseudomonas spp, Proteus spp and Staphylococcus aureus, were the most resistant isolates to the antibiotics, Coagulase-negative Staphylococci was the most sensitive isolate. Out of the ten antibiotics tested ceporex was found to be the least effective with about 100% resistance by the isolates while ciprofloxacin, ceftriaxone, and levofloxacin were the most effective antibiotics with 0% resistance by the bacteria isolates.&#x0D; Conclusion: the study has shown that public surfaces in restaurant can easily be contaminated with a variety of bacterial contaminants that may be multi-drug resistant bacteria strains posing as a possible public health issue.

A Combined Phenotypic-Genotypic Predictive Algorithm for In Vitro Detection of Bicarbonate: β-Lactam Sensitization among Methicillin-Resistant Staphylococcus aureus (MRSA).

Antimicrobial susceptibility testing (AST) is routinely used to establish predictive antibiotic resistance metrics to guide the treatment of bacterial pathogens. Recently, a novel phenotype termed “bicarbonate (NaHCO3)-responsiveness” was identified in a relatively high frequency of clinical MRSA strains, wherein isolates demonstrate in vitro “susceptibility” to standard β-lactams (oxacillin [OXA]; cefazolin [CFZ]) in the presence of NaHCO3, and in vivo susceptibility to these β-lactams in experimental endocarditis models. We investigated whether a targeted phenotypic-genotypic screening of MRSA could rule in or rule out NaHCO3 susceptibility upfront. We studied 30 well-characterized clinical MRSA bloodstream isolates, including 15 MIC-susceptible to CFZ and OXA in NaHCO3-supplemented Mueller–Hinton Broth (MHB); and 15 MIC-resistant to both β-lactams in this media. Using a two-tiered strategy, isolates were first screened by standard disk diffusion for susceptibility to a combination of amoxicillin-clavulanate [AMC]. Isolates then underwent genomic sequence typing: MLST (clonal complex [CC]); agr; SCCmec; and mecA promoter and coding region. The combination of AMC disk susceptibility testing plus mecA and spa genotyping was able to predict MRSA strains that were more or less likely to be NaHCO3-responsive in vitro, with a high degree of sensitivity and specificity. Validation of this screening algorithm was performed in six strains from the overall cohort using an ex vivo model of endocarditis. This ex vivo model recapitulated the in vitro predictions of NaHCO3-responsiveness vs. nonresponsiveness above in five of the six strains.

Diversity of Multidrug-Resistant Bacteria in an Urbanized River: A Case Study of the Potential Risks from Combined Sewage Overflows

Wastewater contamination and urbanization contribute to the spread of antibiotic resistance in aquatic environments. This is a particular concern in areas receiving chronic pollution of untreated waste via combined sewer overflow (CSO) events. The goal of this study was to expand knowledge of CSO impacts, with a specific focus on multidrug resistance. We sampled a CSO-impacted segment of the James River (Virginia, USA) during both clear weather and an active overflow event and compared it to an unimpacted upstream site. Bacteria resistant to ampicillin, streptomycin, and tetracycline were isolated from all samples. Ampicillin resistance was particularly abundant, especially during the CSO event, so these isolates were studied further using disk susceptibility tests to assess multidrug resistance. During a CSO overflow event, 82% of these isolates were resistant to five or more antibiotics, and 44% were resistant to seven or more. The latter statistic contrasts starkly with the upstream reference site, where only 4% of isolates displayed resistance to more than seven antibiotics. DNA sequencing (16S rRNA gene) revealed that ~35% of our isolates were opportunistic pathogens, comprised primarily of the genera Stenotrophomonas, Pseudomonas, and Chryseobacterium. Together, these results demonstrate that CSOs can be a significant source of viable clinically-relevant bacteria to the natural environment and that multidrug resistance is an important understudied component of the environmental spread of antibiotic resistance.

Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1

Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens.

The Evolution of Multidrug Resistance in an Isolated Pseudomonas Strain

As an unintentional result of the extensive use of antibiotics in healthcare and agriculture, antibiotics have become an increasingly prevalent selective pressure on bacteria. This forces bacteria to evolve and acquire antibiotic-resistant genes or mutations in order to survive. Suppose a bacterial strain acquires resistance to three or more antibiotics. In that case, it is deemed multidrug-resistant (MDR), and it becomes a potentially more serious problem to solve in the context of healthcare. This study aims to evaluate the acquisition of resistance to multiple antibiotic drugs by an initially susceptible isolated bacterium from a Minnesota forest environment. The bacterium was found to be Pseudomonas by 16s rRNA gene sequencing. Three antibiotics, neomycin, ciprofloxacin, and imipenem, each from a different drug class, were selected to see if this isolate could become resistant over time and exposure. The bacterial strain developed resistance to the selected antibiotics through a series of sequential exposures to increasing concentrations of each drug in this order. As determined by a disc susceptibility test, the initial isolate acquired resistance to all three selected antibiotics. Single nucleotide polymorphisms (SNPs) between the original isolate and the final resistant strain were identified. These SNPs suggest that mutations to efflux transporters and antibiotic protein targets play a role in acquiring and maintaining antibiotic resistance. KEYWORDS: Multidrug Resistance; Antibiotics; Neomycin; Ciprofloxacin; Imipenem; Pseudomonas; Evolution; MDR; Minnesota Environment

One year period of invasive pneumococcal disease in children from a tertiary care hospital in Turkey in the post-vaccine era

ABSTRACT The incidence of invasive pneumococcal disease (IPD) has decreased after pneumococcal conjugate vaccine used; however, a breakthrough infection may still be seen after vaccination. In this study, eight pediatric inpatients and nine episodes with IPD in our center were included. Their age and gender, diagnoses, facilitating factors, the status of immunization and the antibiotic resistance of Pneumococci, serotypes of Pneumococci were noted. The isolates were subjected to disc susceptibility tests for penicillin, macrolides, and fluoroquinolones, according to the guidelines of the Clinical and Laboratory Standards Institute. Of the vaccinated seven cases, four of them (57.1%) developed IPD which their serogroups were in vaccine content. It was observed that all four cases in question had an underlying facilitating factor. Pneumococcal antibiotic susceptibility is also crucial. Three of nine isolates (33.4%) were resistant, and one isolate (11.2%) was intermediate susceptible to penicillin. Six of the nine isolates (66.7%) had macrolide resistance in our investigation. Invasive pneumococcal infections with serogroups that exist in pneumococcal conjugate vaccine content may occur in vaccinated individuals.

Phenotypic and genotypic detection of ESBL-producing E. coli isolates from chicken skin in Bosnia and Herzegovina.

Aim To identify E. coli from chicken meat, establish their antibiotic resistance profiles and to confirm ESBL isolates with real time PCR, as well as to identify risk factors and farming practice associated with the antimicrobial resistance E. coli. Methods The study included 100 chicken skin samples collected randomly from retail supermarkets, butcheries and slaughterhouses. Disk susceptibility testing was performed using the Kirby-Bauer method. Detection of ESBL-producing isolates was performed with double disk synergy test. Molecular analysis of phenotypic ESBL-producing Escherichia coli strains was performed at 7500 real time PCR System. Molecular-genetic analysis included detection of CTX-M 1, 2, and 9 gene families and mutations in the TEM and SHV encoding extended spectrum β-lactamases. Results Prevalence of the phenotypic ESBL-producing E. coli isolates was 29%, and they exhibited remarkable sensitivity to carbapenems (100%) as well as to amikacin (93.10%). All ESBL-producing strains were multidrug resistant. Molecular analysis was performed as the final confirmation of the production of extended spectrum β - lactamases for 24 isolates out of 29 phenotypicaly ESBL-producing E. coli isolates. Conclusion It is important to pay attention to people's awareness of bacterial antimicrobial resistance in food chain, as well as to understand its effects on human health and the environment. Phenotypic and molecular analysis demonstrated the presence of ESBL-producing E. coli isolates from chicken skin samples.

Area of technical uncertainty for susceptibility testing of amoxicillin/clavulanate against Escherichia coli: analysis of automated system, Etest and disk diffusion methods compared to the broth microdilution reference

ObjectivesThe European Committee on Antimicrobial Susceptibility Testing (EUCAST) recently warned about an area of technical uncertainty (ATU) of amoxicillin/clavulanate (AMX/C) disk susceptibility testing against members of the Enterobacterales. Thus, we aimed to compare the reliability of three routine methods and to evaluate the impact of the ATU. Methods286 Escherichia coli strains (including 159 AMX-resistant strains) were categorized for the two EUCAST AMX/C breakpoints by disk diffusion (Bio-Rad), the Phoenix automated system (Becton Dickinson) and the Etest (AES) compared to the broth microdilution reference method. ResultsBy microdilution, 84.2% of strains were AMX/C-susceptible using the urinary breakpoint (MIC ≤32 mg/L) and 62.2% using the systemic breakpoint (MIC ≤8 mg/L), with 63.6% of MICs between 4 and 16 mg/L. For the systemic breakpoint, category agreement (CA) and very major error (VME) were unacceptable for the Etest (71.7% and 27.3%), disk (73.1% and 23.4% at 19-mm cut-off) and to a lesser extent for the Phoenix system (83.6% and 10.5%). For disks, an unacceptable VME rate was observed for diameters up to 22 mm, probably due to overcharged disks. For the Etest, VMEs were high at 6 mg/L (46/63) and 8 mg/L (22/29). For the urinary breakpoint, CA was more acceptable for disk (88.9%) and Etest (84.3%) but was unevaluable for Phoenix. ConclusionAMX/C susceptibility testing of E. coli for systemic breakpoint was unreliable with the three routine methods, explained mainly by the high prevalence (~60%) of strains with microdilution MICs around the breakpoint (8 mg/L). Our data confirmed the EUCAST 19–20-mm ATU for disk and suggest introducing ATU for Etest MIC values of 6 and 8 mg/L.