Disk Diffusion Antibiotic Susceptibility Testing Research Articles

Improving Turnaround Times for Routine Antimicrobial Sensitivity Testing Following European Committee on Antimicrobial Susceptibility Testing Methodology in Patients with Bacteraemia.

Background/Objectives: Bacteraemia can be fatal without antibiotic intervention. Antibiotic Susceptibility Testing (AST) provides the necessary information for targeted antibiotic therapy; however, the traditional method using disc diffusion can take over two days from a positive blood culture. Inappropriate empiric therapy is associated with increased mortality and increased antibiotic resistance, highlighting the need for more rapid turnaround times for AST. By making changes to an established method, turnaround times can be reduced. Methods: Eighty-two patient positive blood culture samples were collected from January to April 2022, representing the range of common bacteria causing sepsis. This followed the normal methodology in the laboratory of inoculating agar from positive blood cultures in preparation for European Committee on Antimicrobial Susceptibility Testing (EUCAST) disc diffusion AST method. EUCAST methodology outlines that disc diffusion should be performed on isolates from an overnight culture of 16-24 h. This study looked at comparing disc diffusion results from cultures with 6 h of incubation to those with incubation times of 24 h, after organism identification by MALDI-ToF. Results from 6-h and 24-h cultures were compared by disc zone sizes and by interpreted susceptibility reading following EUCAST guidelines of sensitive, resistant, susceptible with increased exposure, or an area of technical uncertainty. Results: A total of 99.65% interpreted susceptibility readings matched across all organisms to all relevant antibiotics, with an average zone size difference of 1.08 mm between results from 6 h versus 24 h cultures. Conclusions: This method offers a non-automated way of using the traditional disc diffusion method, reducing turnaround times while still producing reliable and accurate results. This would mean validated ASTs can be set up in the same day as a blood culture flags positive rather than waiting for a longer culture. As this method is widely used within the laboratory already, it would mean that additional training is not required, as the process is the same, and only incubation time varies. This would positively impact patient outlook due to the shorter use of empiric therapy, and benefit antimicrobial stewardship (AMS).

Prevalence and antimicrobial susceptibility pattern of Vibrio cholerae isolates from cholera outbreak sites in Ethiopia

BackgroundCholera is an acute infectious disease caused by ingestion of contaminated food or water with Vibrio cholerae. Cholera remains a global threat to public health and an indicator of inequity and lack of social development. The aim of this study was to assess the prevalence and antimicrobial susceptibility pattern of V. cholerae from cholera outbreak sites in Ethiopia.MethodsAcross-sectional study was conducted from May 2022 to October 2023 across different regions in Ethiopia: Oromia National Regional State, Amhara National Regional State and Addis Ababa City Administration. A total of 415 fecal samples were collected from the three regions. Two milliliter fecal samples were collected from each study participants. The collected samples were cultured on Blood Agar, MacConkey Agar and Thiosulfate Citrate Bile Salt Sucrose Agar. A series of biochemical tests Oxidase test, String test, Motility, Indole, Citrate, Gas production, H2S production, Urease test were used to identify V. cholerae species. Both polyvalent and monovalent antisera were used for agglutination tests to identify and differentiate V. cholerae serogroup and serotypes. In addition, Kirby-Bauer Disk diffusion antibiotic susceptibility test method was done. Data were registered in epi-enfo version 7 and analyzed by Statistical Package for Social Science version 25. Descriptive statistics were used to determine the prevalence of Vibrio cholerae. Logistic regression model was fitted and p-value < 0.05 was considered as statically significant.ResultsThe prevalence of V. cholerae in the fecal samples was 30.1%. Majority of the isolates were from Oromia National Regional State 43.2% (n = 54) followed by Amhara National Regional State 31.2% (n = 39) and Addis Ababa City Administration 25.6% (n = 32). Most of the V. cholerae isolates were O1 serogroups 90.4% (n = 113) and Ogawa serotypes 86.4% (n = 108). Majority of the isolates were susceptible to ciprofloxacin 100% (n = 125), tetracycline 72% (n = 90) and gentamycin 68% (n = 85). More than half of the isolates were resistant to trimethoprim-sulfamethoxazole 62.4% (n = 78) and ampicillin 56.8% (n = 71). In this study, participants unable to read and write were about four times more at risk for V. cholerae infection (AOR: 3.8, 95% CI: 1.07–13.33). In addition, consumption of river water were about three times more at risk for V. cholerae infection (AOR: 2.8, 95% CI: 1.08–7.08).Conclusionour study revealed a high prevalence of V. cholerae from fecal samples. The predominant serogroups and serotypes were O1 and Ogawa, respectively. Fortunately, the isolates showed susceptible to most tested antibiotics. Drinking water from river were the identified associated risk factor for V. cholerae infection. Protecting the community from drinking of river water and provision of safe and treated water could reduce cholera outbreaks in the study areas.

STUDY OF BACTERIAL PATHOGENS CAUSING URINARY TRACT INFECTION AND ANTIBIOGRAM PATTERN IN SYMPTOMATIC ADULTS

Objectives: Urinary tract infections (UTIs) are most common infection seen in community and hospital. It is important to know the causative agent and antibiotic susceptibility pattern to administer specific treatment and prevent drug resistance. Hence, the present study was conducted to know the most common etiological agent and antibiogram of symptomatic UTIs. Methods: The study was conducted at a tertiary care hospital. All the adult patients presented with symptoms of UTI were involved. As per the standard guidelines, clean catch mid-stream urine sample was collected and inoculated on MacConkey’s agar and blood agar by standard loop technique. The isolates with significant bacteriuria were subjected for Kirby-Bauers’ disk diffusion antibiotic susceptibility testing. Results: One thousand two hundred and fifty symptomatic patients were screened for UTI. A total of 358 organisms were isolated in culture with significant bacteriuria. Among 358, Escherichia coli was commonest organism with 267 (74.5%), followed by Klebsiella species accounting for 52 isolates (14.5%) and Citrobacter species 25 (7.0%), and Pseudomonas aeruginosa 14 (4%). Higher degree of sensitivity was seen with imipenem (90%), amikacin (80%), and gentamycin (78%) followed by nitrofurantoin (75%) for E. coli. Lower degree of sensitivity was noted for norfloxacin (50%), ceftriaxone (62%), cotrimoxazole (56%), and pipericillin-tazobactum (60%). Klebsiella was sensitive to amikacin (74%) and imipenem (80%). Conclusion: In the present study, E. coli was the most common organism causing UTI. Nitrofurantoin can be started as empirical treatment for lower UTI. Gentamycin was found to be equally effective as amikacin so that it can be used instead of amikacin to decrease the selective pressure on amikacin.

In vitro phytochemical analysis and antibacterial and antifungal efficacy assessment of ethanolic and aqueous extracts of Rumex nervosus leaves against selected bacteria and fungi.

Scientists are interested in identifying natural antibiotic substitutes that are effective against drug-resistant pathogenic microbes and spoilage fungi to counter pathogens and reduce the major public health problem of antibiotic residues in animal products. This study aimed to evaluate the antimicrobial activity of Rumex nervosus leaves (RNLs) as a medicinal herb against four bacterial and two fungal strains using absolute ethanol, 50% ethanol, and aqueous extracts. The antimicrobial activities of various RNL extracts against selected microbes were evaluated using the disk diffusion antibiotic susceptibility test, minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), minimum fungicidal concentrations, and the poisoned food technique. The absolute ethanol RNL extract showed the best bacteriostatic/bactericidal activity against Salmonella Typhimurium, Escherichia coli, and Staphylococcus aureus (MIC/MBC: 0.20/0.40, 0.20/0.40, and 0.32/0.65 mg/mL, respectively). The diameter of the zone of inhibition was larger (p < 0.05) for the 100% ethanol RNL extract (8.17 mm) against Salmonella Typhimurium, the 50% ethanol-RNL extract (11.5 mm) against E. coli, and the aqueous RNL extract (14.0 mm) against S. aureus than for any other bacterial isolate. The aqueous RNL extract strongly (p < 0.0001) inhibited the mycelial growth of Aspergillus fumigatus (100%) and Aspergillus niger (81.4%) compared with the control. The results of this study suggest that RNL is a promising new natural antimicrobial agent for food preservation. To date, most research on the antimicrobial properties of natural herbs has been conducted in vitro, with few exceptions in vivo and intervention-based research.

Impact of the introduction of EUCAST's concept of "area of technical uncertainty".

On the first of January 2019, the European Committee on Antimicrobial Susceptibility Testing, EUCAST, introduced the concept of "area of technical uncertainty" (ATU). The aim was to report on the incidence of ATU test results in a selection of common bacterial species and the subsequent impact on antimicrobial resistance categorization and workload. A retrospective analysis of clinical samples collected from February 2019 until November 2019 was performed. Susceptibility to amoxicillin-clavulanic acid and piperacillin-tazobactam in Enterobacterales (Escherichia spp., Klebsiella spp., Proteus spp.), piperacillin-tazobactam in Pseudomonas aeruginosa, and amoxicillin-clavulanic acid and cefuroxime in Haemophilus influenzae was studied. Disk diffusion antibiotic susceptibility testing was read and interpreted by ADAGIO 93400 automated system (Bio-Rad, France). In case of an inhibition zone in the ATU, strains were retested using gradient minimal inhibitory concentration method (Etest, BioMérieux, France). Overall, 14,164 isolate-antibiotic combinations were tested in 7922 isolates, resulting in 1204 (8.5%) disk zone diameters in the ATU region. Retesting of ATUs with Etest resulted in a category change from S to R for amoxicillin-clavulanic acid in 63/498 (12.7%) of Escherichia spp., 2/58 (3.4%) of Klebsiella spp., 2/37 (5.4%) of Proteus spp., and 6/125 (4.8%) of Haemophilus influenzae. For piperacillin-tazobactam, a category change from S to R was found in 33/92 (35.9%) of Pseudomonas aeruginosa. We conclude that ATU testing has a substantial impact on the correct interpretation of antimicrobial resistance, at the expense of turn-around time and with the cost of additional workload.

Identity and Antibiogram of Bacterial Isolates from Owerri Modern Abattoir, Imo State, Nigeria

Aim: The identity and antibiogram of bacterial isolates from Owerri modern abattoir in Imo State, Nigeria, was investigated with the aim of determining the bacterial profile of the abattoir and their susceptibility pattern to commonly used antibiotics.&#x0D; Study Design: Cross sectional study&#x0D; Place and Duration of Study: This study was carried out in Owerri Modern Abattoir located within Owerri metropolis from June to November, 2020.&#x0D; Methodology: Questionnaires were used to obtain participants’ consent, demographic data and sanitary practices in the abattoir. Samples were taken and bacteriological analysis of the samples done using pour plate method. Disc diffusion antibiotic susceptibility testing and minimum inhibitory concentration were performed after colony counting, identification and characterization of the isolates using standard microbiological and biochemical techniques.&#x0D; Results: Mean viable bacterial counts were generally high with highest counts from contaminated soil (6.13x106CFU/ml) and least from workers hands (1.17x106CFU/ml). Escherichia coli had the highest prevalence (18.0%), with the highest counts from soil (3.10%). Vibrio cholerae recorded the least prevalence (0.62%), and was isolated only from washing water. High resistance to antibiotics was observed.&#x0D; Conclusion: Government authority and the general public are advised to ensure adequate environmental sanitation and proper cooking of meat before consumption in order to mitigate the incidence of infection and antibiotic resistance.

Rapid and real-time monitoring of bacterial growth against antibiotics in solid growth medium using a contactless planar microwave resonator sensor

Infection diagnosis and antibiotic susceptibility testing (AST) are pertinent clinical microbiology practices that are in dire need of improvement, due to the inadequacy of current standards in early detection of bacterial response to antibiotics and affordability of contemporarily used methods. This paper presents a novel way to conduct AST which hybridizes disk diffusion AST with microwave resonators for rapid, contactless, and non-invasive sensing and monitoring. In this research, the effect of antibiotic (erythromycin) concentrations on test bacterium, Escherichia coli (E. coli) cultured on solid agar medium (MH agar) are monitored through employing a microwave split-ring resonator. A one-port microwave resonator operating at a 1.76 GHz resonant frequency, featuring a 5 mm2 sensitive sensing region, was designed and optimized to perform this. Upon introducing uninhibited growth of the bacteria, the sensor measured 0.005 dB/hr, with a maximum change of 0.07 dB over the course of 15 hours. The amplitude change decreased to negligible values to signify inhibited growth of the bacteria at higher concentrations of antibiotics, such as a change of 0.005 dB in resonant amplitude variation while using 45 µg of antibiotic. Moreover, this sensor demonstrated decisive results of antibiotic susceptibility in under 6 hours and shows great promise to expand automation to the intricate AST workflow in clinical settings, while providing rapid, sensitive, and non-invasive detection capabilities.

AI-based mobile application to fight antibiotic resistance

Antimicrobial resistance is a major global health threat and its development is promoted by antibiotic misuse. While disk diffusion antibiotic susceptibility testing (AST, also called antibiogram) is broadly used to test for antibiotic resistance in bacterial infections, it faces strong criticism because of inter-operator variability and the complexity of interpretative reading. Automatic reading systems address these issues, but are not always adapted or available to resource-limited settings. We present an artificial intelligence (AI)-based, offline smartphone application for antibiogram analysis. The application captures images with the phone’s camera, and the user is guided throughout the analysis on the same device by a user-friendly graphical interface. An embedded expert system validates the coherence of the antibiogram data and provides interpreted results. The fully automatic measurement procedure of our application’s reading system achieves an overall agreement of 90% on susceptibility categorization against a hospital-standard automatic system and 98% against manual measurement (gold standard), with reduced inter-operator variability. The application’s performance showed that the automatic reading of antibiotic resistance testing is entirely feasible on a smartphone. Moreover our application is suited for resource-limited settings, and therefore has the potential to significantly increase patients’ access to AST worldwide.

Bacterial Multidrug Resistant Patterns Associated with Intensive Care Unit Infections in Akum, Cameroon

Background: Patients admitted into the intensive care unit (ICU) usually have impaired immunity and are therefore at high risk of acquiring hospital associated infections. Infections caused by multidrug resistant bacteria now constitute a major problem, limiting the choice of antimicrobial therapy.&#x0D; Objectives: This study was aimed at determining the antimicrobial resistance pattern of pathogens causing ICU infections at the Catholic Holy Family Hospital Akum-Bamenda. The etiological agents, prevalence and types ICU infections were also determined.&#x0D; Methods: Thirteen months hospital associated infections surveillance was conducted in the hospital for patients admitted into ICU. Blood, urine, tracheal aspirate and wound biopsies specimens were collected under strict asepsis and sent to the Medical Microbiology laboratory of the same institution for immediate processing. All pathogens were isolated and identified by standard microbiological methods. Disk diffusion antibiotic susceptibility testing was performed and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines.&#x0D; Results: The overall prevalence of ICU infections was 30.9% out of which 12.9% were bloodstream infections, 31.5% urinary tract infections, 38.9% pneumonia, and 16.7% skin and soft tissue infections. Klebsiella species and Escherichia coli were the predominant pathogens. Multidrug resistant organisms constituted 59.3% of the pathogens, MDR Klebsiella spp and MDR E. coli were 70.8% and 71.4% respectively. Resistance to Cefuroxime was the highest (92.9%) while Meropenem had the least resistance (21.4%).&#x0D; Conclusion: There is a high prevalence of multidrug resistant bacteria causing ICU infections. With the advent of covid-19, infection control procedures and institution of functional antimicrobial stewardship are needed to be put in place in order to cover come this herculean task.

Extended Spectrum Beta Lactamase (ESBL), blaTEM,blaSHV and blaCTX-M, Resistance Genes in Community and Healthcare Associated Gram Negative Bacteria from Osun State, Nigeria.

Extended Spectrum Beta Lactamase (ESBL) production in gram negative bacteria confers multiple antibiotic resistance, adversely affecting antimicrobial therapy in infected individuals. ESBLs result from mutations in β-lactamases encoded mainly by the blaTEM,blaSHV and blaCTX-M genes. The prevalence of ESBL producing bacteria has been on the increase globally, especially its upsurge among isolates from community-acquired infections has been observed. To determine ESBL prevalence and identify ESBL genes among clinical isolates in Osun State, Nigeria. A cross-sectional study was carried out from August 2016 - July 2017 in Osun State, Nigeria. Three hundred and sixty Gram-negative bacteria recovered from clinical samples obtained from both community and healthcare-associated infections were tested. They included 147 Escherichia coli (40.8%), 116 Klebsiella spp (32.2%), 44 Pseudomonas aeruginosa (12.2%) and 23 Proteus vulgaris (6.4%) isolates. Others were Acinetobacter baumannii, Serratia rubidae, Citrobacter spp, Enterobacter spp and Salmonella typhi. Disk diffusion antibiotic susceptibility testing was carried out, isolates were screened for ESBL production and confirmed using standard laboratory procedures. ESBLs resistance genes were identified by Polymerase Chain Reaction (PCR). All isolates demonstrated multiple antibiotic resistance. Resistance to ampicillin, amoxicillin with clavulanate and erythromycin was 100%, whereas resistance to Imipenem was very low (5.0%). The overall prevalence of ESBL producers was 41.4% with Klebsiella spp as the highest ESBL producing Enterobacteriacaea. ESBL producers were more prevalent among the hospital pathogens than community pathogens, 58% vs. 29.5% (p=0.003). ESBL genes were detected in all ESBL producers with the blaCTX-M gene predominating (47.0%) followed by blaTEM (30.9%) and blaSHV gene was the least, 22.1%. The blaCTX-M gene was also the most prevalent in the healthcare pathogens (62%) but it accounted for only 25% in those of community origin. A high prevalence of ESBL producing gram-negative organisms occurs both in healthcare and in the community in our environment with the CTX-M variant predominating. Efforts to control the spread of these pathogens should be addressed.

A new 5-plex PCR detection method for acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains

Acute hepatopancreatic necrosis disease (AHPND) is a shrimp disease caused by a unique strain of Vibrio parahaemolyticus (VpAHPND). This strain contains a highly pathogenic ~69 kbp (pVA1) plasmid encoding PirAvp and/or PirBvp deadly toxins. The pVA1-like plasmid is also reported to be found in Vibrio harveyi, Vibrio campbellii and Vibrio owensii. Therefore, prompt detection of the bacterial species carrying the PirAvp and/or PirBvp toxins producing genes is essential to control the spread of the disease. In this study, a new 5-plex PCR detection method was developed and optimised to detect VpAHPND or tetracycline resistant VpAHPND strain. This technique is designed to include five pairs of primers, which allow a comprehensive PCR diagnostic method for AHPND. The primers flanked five regions which are the Photorhabdus insect-related (Pir) toxin-like genes; pirAvp and pirBvp, thermolabile hemolysin (tlh), housekeeping (toxR) or tetracycline resistance (tetB) genes and also 16S ribosomal RNA (16s rRNA) gene as an internal amplification control (IAC). The detection limit of the 5-plex PCR method is at 10 pg/μl of extracted genomic DNA. Whereas, the specificity of the method analysed using nine VpAHPND strains, three non-AHPND Vibrio species and ten other bacterial species revealed that the method does not give unspecific reactions with non-AHPND bacteria. The 5-plex PCR method evaluated using three locally isolated VpAHPND strains and 41 isolates of Vibrio spp. obtained from field samples confirmed that the method developed allows rapid, sensitive and reliable detection of AHPND-causing bacterial strains. The VpAHPND strains that were detected positive for the tetB gene also showed resistant to tetracycline by disk diffusion antibiotic susceptibility test. The presence of IAC and the primers targeting the tetB gene helps to indicate false-negative results and to detect tetracycline resistant AHPND strains respectively thus differentiating this method from previously reported AHPND PCR methods.

Effect of initial bacterial innoculum density on zones of inhibition in disk diffusion antibiotic susceptibility testing

Effect of initial bacterial innoculum density on zones of inhibition in disk diffusion antibiotic susceptibility testing

Vancomycin resistant enterococcal (VRE) colonization among patients treated in intensive care units at the National Hospital of Sri Lanka, and determination of genotype/s responsible for resistance

The aim of this study was to assess the epidemiology of VRE colonization among patients in the intensive care units (ICU) of the National Hospital of Sri Lanka (NHSL). A cross sectional study was carried out on 218 patients admitted to 12 ICUs of the NHSL from January to March 2012. Rectal swabs were collected on day 0, 4, 8 and every 4th day thereafter till discharge. Enterococci were isolated on selective media and identified up to species level using standard bacteriological procedures. Standardized disc diffusion antibiotic susceptibility testing to ampicillin, teicoplanin and vancomycin was performed using the Clinical and Laboratory Standards Institute (CLSI) method. Minimum inhibitory concentrations to vancomycin were determined, using the E-test in strains showing intermediate or frank resistance to vancomycin by disc diffusion. Genotype determination (van A / van B) was carried out on isolates identified as VRE using the polymerase chain reaction. Patients positive for VRE colonization were followed up to discharge or death. VRE prevalence in the study sample was 5%. Univariate analysis showed that the use of metronidazole (odds ratio [OR] :15.73;95% 95% confidence interval [CI] : 3.94-62.67,P<0.05) or teicoplanin (OR: 12.56; 95% CI:2.65 – 59.52, p< 0.05) and diabetes (OR: 05.13; 95% CI: 1.36 – 18.7, p< 0.05) or hemodialysis during ICU stay (OR: 7.38 ;95% CI : 1.69-32.16, P<0.05) were associated with an increased risk of VRE colonization. The 5% prevalence of VRE colonization detected signals the emergence of VRE in the intensive care setting in Sri Lanka.

Prevalence of MDR Pseudomonas aeruginosa of war-related wound and burn ward infections from some conflict areas of Western Yemen

Abstract Background and objectives The antibiotic resistant Pseudomonas aeruginosa affecting war-related wound injuries is a serious problem with regional and global implications, which requires antibiotic susceptibility testing on a regular basis. The primary aim of this study was to determine the prevalence for multi-drug resistance Pseudomonas aeruginosa of war-related wound infections from conflict areas in Hodiedah, Western Yemen. Methods and materials A study was undertaken with ninety-nine wound swabs from war-related wound infections patients those were hospitalized to Wound Burn Centre at Organization The General AL-Thawra Hospital, Hodiedah Western Yemen. The isolates were cultured and identified by standard microbiological techniques and with the use of Kirby- Bauer disc diffusion antibiotic susceptibility testing was done. Results Out of 99 swab isolates 46 were P. aeruginosa (46.46%). Highest incidence of 63.1% was found in the age group of 20–40 years. Isolates exhibited resistance to aminoglycosides; Gentamycin (87%), Tobramycin (78.2%) and Amikacin (82.6%). Curiously, 65.2% P. aeruginosa isolates were multidrug resistance (MDR). Conclusion This is the first report documenting enhanced MDR P. aeruginosa from war-related wound infections from Yemen. The sampling, timing of medical intervention plays crucial role in preventing nosocomial transmission of MDR P. aeruginosa strains.

Antibiotic Susceptibility Profile of Bacteria Isolated from Door Handles of University of Benin Teaching Hospital, Benin City, Edo State, Nigeria

In recent times, surfaces of hospital environment, constantly exposed to activities of patients and health-care workers, have been identified as potential routes for horizontal dissemination of drug resistant microorganisms of public health significance. This study was conducted to investigate the antibiotic resistance profile of bacteria isolated from samples collected from door handles of various units in University of Benin Teaching Hospital (UBTH), Benin City, Edo State, Nigeria. Out of the 66 door handles sampled and analysed bacteriologically, 68 predominant bacteria distributed among five (5) species of Escherichia coli (30.9%), Staphylococcusaureus (26.5%), Pseudomonas aeruginosa (17.6%), Streptococcus pneumonia (13.2%) and Bacillus subtilis (11.8%) were isolated. Kirby Bauer techniques of disk diffusion antibiotic susceptibility test on the isolates using 9 commercial antibiotics according to Clinical Laboratory Standards Institute (CLSI) procedure showed resistance of P. aeruginosa to five (5) drugs (Sulfamethoxazole-Trimethoprim, Streptomycin, Ampicillin, Cephalexin and Nalidixic acid), E. coli to five (5) drugs (Ofloxacin, Sulfamethoxazole-Trimethoprim, Ampicillin, Cephalexin and Nalidixic acid), S. pneumoniaeto two drugs (Ampicilin and Nalidixic acid) and S aureus to only Nalidixic acid. Agarose gel electrophoresis carried out on their plasmid DNA revealed detectable fragments in P. aeruginosa and E. coli. After plasmid curing, the isolates became sensitive to Streptomycin, Cephalexin,Ofloxacin, Sulfamethoxazole-Trimethoprim, Ampicillin and Ciprofloxacin. Findings from this study suggest the presence of plasmid mediated multi-drug resistant P. aeruginosa and E. coli on surfaces of hospital door handles. Hence, public education on proper personal hygiene and disinfection techniques are recommended in hospitals.

Rapid disc diffusion antibiotic susceptibility testing for Pseudomonas aeruginosa, Acinetobacter baumannii and Enterococcus spp.

BackgroundWe investigated the feasibility of rapid disc diffusion antibiotic susceptibility testing (rAST) with reading of inhibition zones after 6 and/or 8 h of incubation for Enterococcus faecalis, Enterococcus faecium, Pseudomonas aeruginosa and Acinetobacter baumannii. In addition, we evaluated discrimination of resistant populations from the WT populations at early timepoints and the requirement for clinical breakpoint adaptations for proper interpretation of rAST data.MethodsIn total, 815 clinical strains [E. faecalis (n = 135), E. faecium (n = 227), P. aeruginosa (n = 295) and A. baumannii (n = 158)] were included in this study. Disc diffusion plates were streaked, incubated and imaged using the WASPLabTM automation system. WT populations and non-WT populations were defined using epidemiological cut-offs.Results and conclusionsrAST at 6 and 8 h was possible for A. baumannii and enterococci with readability of inhibition zones >90%. Overall categorical agreement of rAST at 6 h with AST at 18 h was 97.2%, 97.4% and 95.3% for E. faecalis, E. faecium and A. baumannii, respectively. With few exceptions, major categorization error rates were <1% for A. baumannii, and vancomycin-resistant E. faecium were clearly separated from the WT at 6 h. For P. aeruginosa the average readability of inhibition zones was 68.9% at 8 h and we found an overall categorical agreement of 94.8%. Adaptations of clinical breakpoints and/or introduction of technical buffer zones, preferably based on aggregated population data from various epidemiological settings, are required for proper interpretation of rAST.

Rapid detection of ESBL, carbapenemases, MRSA and other important resistance phenotypes within 6-8 h by automated disc diffusion antibiotic susceptibility testing.

In principle, automated systems allow rapid reading of disc diffusion AST (rAST) within 6-8 h. This study analysed whether rAST can discriminate resistance phenotypes such as ESBL, carbapenemases and MRSA/methicillin-resistant Staphylococcus epidermidis from WT populations. We describe species-drug combinations that may require clinical breakpoint adaptions for early reading due to zone diameter changes during the incubation period. In total, 1852 clinical strains [Escherichia coli (n = 475), Klebsiella pneumoniae (n = 375), Enterobacter cloacae (n = 301), Staphylococcus aureus (n = 407) and S. epidermidis (n = 294)] were included in this study comprising WT populations and important resistance phenotypes, e.g. ESBL, carbapenemases and MRSA. We assessed (i) separation of resistance phenotypes and WT populations after 6, 8 and 12 h as compared with the 18 h standard, and (ii) diameter changes of WT populations and associated putative epidemiological cut-offs during the incubation period. Disc diffusion plates were automatically streaked, incubated and imaged using the WASPLabTM system. We demonstrated that important resistance phenotypes could reliably be separated from WT populations at early reading times for the most prevalent bacterial pathogens encountered in the clinical laboratory. Current AST expert rules and algorithms for identification of resistance mechanisms can readily be applied for rAST, e.g. EUCAST recommended rules for detection of ESBL, AmpC, carbapenemases and MRSA/methicillin-resistant S. epidermidis. However, several species-drug combinations may require clinical breakpoint adaptations when using rAST as the diameter, and hence the epidemiological cut-off, changes during the incubation period.

Nasal carriage of methicillin resistant Staphylococcus aureus among health care workers at Al Shifa hospital in Gaza Strip

BackgroundNasal carriage of Staphylococcus aureus among hospital personnel is a common cause of hospital acquired infections. Emergence of drug resistant strains especially methicillin resistant S. aureus (MRSA) is a serious problem in hospital environment. Therefore, the aim of this study was to determine the nasal carriage rate of S. aureus and MRSA among Health Care Workers (HCWs) at Al Shifa Hospital, the major hospital in Gaza Strip.MethodsA cross sectional study was conducted on 200 HCWs. Nasal swabs were collected during February — April 2015, and cultured on blood and mannitol salt agar. The isolates were identified as S. aureus based on morphology, coagulase test, DNase test and mannitol salt agar fermentation. Disk diffusion antibiotic susceptibility tests were performed according to the guidelines of the Clinical and Laboratory Standards Institute. MRSA were confirmed by detection of the mecA gene by PCR.ResultsOut of the 200 healthcare workers, 62 (31%) carried S. aureus, of which 51 (82.3%) were MRSA. Therefore, 25.5% of all HCWs were identified as MRSA carriers. MRSA carriage rate was highest among nurses (30.4%) whereas the carriage rate among doctors was (16%). The majority of MRSA carriers were workers of internal medicine department and surgical wards (41.3 and 35% respectively). Out of the 51 MRSA isolates identified by oxacillin disc resistance, 40 were confirmed by PCR targeting the mecA gene. Penicillin showed the highest rate of resistance among MRSA and MSSA isolates (100%).ConclusionsThe high rate of nasal MRSA carriage among healthcare workers found in this study is alarming and highlights the need for adjusted infection control measures to prevent MRSA transmission from HCWs to the vulnerable patient.

Novel Isoflavone from the Cockroach Periplaneta americana

A novel compound, 13,13-dimethyl,12-(16-hydroxy,16,16-dimethyl)-propanol-∆11,12-hydrogenated pyranyl-7,8[benzo]-4′-methoxyisoflavone (1), together with two known insect secondary metabolites 2 and 3, was isolated from the 70% methanol extract of Periplaneta americana cockroaches. The structures of the isolated compounds were established by NMR spectroscopic and HR-ESI-MS analysis. Their antibacterial activities were determined by Kirby-Bauer disc diffusion antibiotic susceptibility testing. The results indicated that both isoflavone compounds 1 and 2 showed significant inhibitory effects against the Gram-positive bacterium B. subtilis with minimum inhibitory concentrations (MIC) values of 15 μg/mL.

Optimized In Vitro Antibiotic Susceptibility Testing Method for Small-Colony Variant Staphylococcus aureus.

Staphylococcus aureus small-colony variants (SCVs) emerge frequently during chronic infections and are often associated with worse disease outcomes. There are no standardized methods for SCV antibiotic susceptibility testing (AST) due to poor growth and reversion to normal-colony (NC) phenotypes on standard media. We sought to identify reproducible methods for AST of S. aureus SCVs and to determine whether SCV susceptibilities can be predicted on the basis of treatment history, SCV biochemical type (auxotrophy), or the susceptibilities of isogenic NC coisolates. We tested the growth and stability of SCV isolates on 11 agar media, selecting for AST 2 media that yielded optimal SCV growth and the lowest rates of reversion to NC phenotypes. We then performed disk diffusion AST on 86 S. aureus SCVs and 28 isogenic NCs and Etest for a subset of 26 SCVs and 24 isogenic NCs. Growth and reversion were optimal on brain heart infusion agar and Mueller-Hinton agar supplemented with compounds for which most clinical SCVs are auxotrophic: hemin, menadione, and thymidine. SCVs were typically nonsusceptible to either trimethoprim-sulfamethoxazole or aminoglycosides, in accordance with the auxotrophy type. In contrast, SCVs were variably nonsusceptible to fluoroquinolones, macrolides, lincosamides, fusidic acid, and rifampin; mecA-positive SCVs were invariably resistant to cefoxitin. All isolates (both SCVs and NCs) were susceptible to quinupristin-dalfopristin, vancomycin, minocycline, linezolid, chloramphenicol, and tigecycline. Analysis of SCV auxotrophy type, isogenic NC antibiograms, and antibiotic treatment history had limited utility in predicting SCV susceptibilities. With clinical correlation, this AST method and these results may prove useful in directing treatment for SCV infections.