Routine Antimicrobial Susceptibility Testing Research Articles

A simplified pyrazinamidase test for Mycobacterium tuberculosis pyrazinamide antimicrobial susceptibility testing

ABSTRACT Pyrazinamide (PZA) is an important first-line drug for tuberculosis (TB) treatment by eradicating the persisting Mycobacterium tuberculosis complex (MTBC). Due to cost and technical challenges, end TB strategies are hampered by the lack of a simple and reliable culture-based PZA antimicrobial susceptibility testing (AST) for routine use. We initially developed a simplified chromogenic pyrazinamidase (PZase) test in the TB reference laboratory using a training set MTBC isolates with various drug-resistant profiles, and validated its performance using consecutive BACTEC MGIT 960 (MGIT)-culture-positive culture in 10 clinical laboratories. The pncA gene Sanger sequencing results were used as the reference, and compared to the MGIT-PZA AST. Differential diagnosis of Mycobacterium bovis was conducted using patented in-house real-time PCR. Of the 106 training isolates, the PZase test and MGIT-PZA AST showed 100.0% and 99.1% concordance as compared to Sanger sequencing, respectively. We found 32.1% (34/106) isolates harbored pncA mutations, including one isolate with silent mutation S65S. For validation, 1,793 clinical isolates were tested including 150 duplicate isolates from specimens of the same cases and 16 isolates with uncharacterized drug resistance (UDR)-associated mutations. Excluding duplicated and UDR isolates, we identified 2.6% (43/1,627) PZA-resistant isolates, including 1.3% (21/1,627) M . bovis isolates. The kappa values were 0.851–1.000. In addition, the accuracy of the PZase test conducted by 10 laboratories was 98.5%–100.0%. Our simplified PZase test demonstrated high concordance with Sanger sequencing and MGIT-PZA AST. Integrating the PZase test into routine first-line AST is effortless and represents an improvement in laboratory services for ending TB. IMPORTANCE We developed and validated a simple pyrazinamidase (PZase) test for pyrazinamide (PZA) antimicrobial susceptibility testing (AST). Our results demonstrated that the PZase test had high agreement with the pncA gene sequencing and MGIT-PZA AST. Integrating PZase test into routine AST is effortless and represents an improvement in laboratory services for ending TB.

Overall status of carbapenem resistance among clinical isolates of Acinetobacter baumannii: a systematic review and meta-analysis.

Resistance to carbapenems, the first-line treatment for infections caused by Acinetobacter baumannii, is increasing throughout the world. The aim of the present study was to determine the global status of resistance to carbapenems in clinical isolates of this pathogen, worldwide. Electronic databases were searched using the appropriate keywords, including: 'Acinetobacter' 'baumannii', 'Acinetobacter baumannii' and 'A. baumannii', 'resistance', 'antibiotic resistance', 'antibiotic susceptibility', 'antimicrobial resistance', 'antimicrobial susceptibility', 'carbapenem', 'carbapenems', 'imipenem', 'meropenem' and 'doripenem'. Finally, following some exclusions, 177 studies from various countries were included in this study. The data were then subjected to a meta-analysis. The average resistance rate of A. baumannii to imipenem, meropenem and doripenem was 44.7%, 59.4% and 72.7%, respectively. A high level of heterogeneity (I2 > 50%, P value < 0.05) was detected in the studies representing resistance to imipenem, meropenem and doripenem in A. baumannii isolates. Begg's and Egger's tests did not indicate publication bias (P value > 0.05). The findings of the current study indicate that the overall resistance to carbapenems in clinical isolates of A. baumannii is relatively high and prevalent throughout the world. Moreover, time trend analysis showed that the resistance has increased from the year 2000 to 2023. This emphasizes the importance of conducting routine antimicrobial susceptibility testing before selecting a course of treatment, as well as monitoring and controlling antibiotic resistance patterns in A. baumannii strains, and seeking novel treatment options to lessen the emergence and spread of resistant strains and to reduce the treatment failure.

LABORATORY DETECTION OF COLISTIN-RESISTANT &lt;i&gt;ENTEROBACTERALES&lt;/i&gt; IN TANDEM WITH ROUTINE ANTIBIOGRAM

Since the emergence and spread of carbapenemase-producing Enterobacterales, particularly those carrying metallo-β-lactamases with 16S rRNA methyltransferases for which newly introduced antibiotics are inactive, colistin is a last resort therapy for life-threatening extensively drug-resistant infections. This requires that laboratories use an accurate and reliable method for routine colistin susceptibility testing. The aim of this study was to evaluate the performance and applicability of a colistin screening medium for detection of colistin-resistant isolates simultaneously with routine susceptibility testing. It was evaluated with fifty colistin-resistant and the same number of colistin-susceptible comparator isolates. Our results showed that all colistin-resistant isolates grew on DiaPlate™ EMB Agar + Colistin medium within the standard antibiogram period. In contrast, no growth was observed among the colistin susceptible comparators. DiaPlate™ EMB Agar + Colistin is a screening medium that can detect colistin- resistant Enterobacterales isolates when pure bacterial cultures are tested. As this method for detecting colistin-resistant isolates uses the same inoculum as the Kirby-Bauer method, the screening test for colistin resistance can be conveniently performed on clinical isolates along with routine antimicrobial susceptibility testing.

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database

ObjectivesTo evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. MethodsDNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). ResultsWe processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0–91.9 %) and 81.1 % (95%CI 68.5–93.7 %), while the specificity was 96.7 % (95%CI 90.2–100.0 %) and 56.3 % (95%CI 31.9–80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17–245.5) and 99 min (IQR 31.25–269.75) after sequencing commencement, respectively. ConclusionsNanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

Colistin, the last resort antibiotic: challenges in the implementation of its routine susceptibility testing

Background: colistin has become a critical antibiotic for lifethreatening multidrug resistance Gram-negative infections, particularly carbapenemase-producing bacteria. Detecting colistin resistance in routine microbiology laboratories is crucial for combating these fatal infections poses a challenge. Especially in developing countries, there is a need for a cost-effective, rapid, and user-friendly diagnostic method. Objective: implementing the various available methods for colistin testing is a significant challenge in resource-limited settings due to logistic difficulties and the need for technical expertise. Materials and Methods: this study shares experiences and insights gained while implementing in-vitro colistin susceptibility testing in a high-load bacteriology laboratory of a tertiary care center in Delhi, India. The following test methods for colistin susceptibility testing were incorporated in the routine antimicrobial susceptibility testing of our laboratory: Colistin Agar Test, Colistin Broth Disk Elution Test, Broth Microdilution susceptibility testing. Results: inconsistent growth patterns were observed in the colistin agar dilution Minimum Inhibitory Concentration (MIC) method, which could be resolved only after the preparation of fresh plates containing that specific concentration of colistin. The contamination issue of plates on use over a few days was addressed by pouring agar containing various concentrations of colistin in cottonplugged glass tubes. In the colistin broth disk elution test, due to the non-availability of screw-capped 10 mL glass tubes, MacCornety bottles (30 mL) were used. Subcultures were performed from the turbid wells to rule out the growth of contaminants when encountering discordant MIC values or skipped wells on the colistin broth microdilution test. Conclusions: despite several technical issues in in-vitro colistin susceptibility testing, we have successfully implemented it in our laboratory. Our experiences can offer guidance to laboratories that are still in the process of implementing it.

Cefiderocol – An effective antimicrobial for MDR infections but a challenge for routine antimicrobial susceptibility testing

PurposeCefiderocol is a novel cephalosporin–siderophore conjugate antibiotic that holds promise to thwart infections caused by multi-drug-resistant gram-negative bacilli. Its antibacterial activity against normally susceptible species is not affected by most β-lactamases, including metallo-β-lactamases. Due to the siderophore-mediated entry into the cell, the activity of cefiderocol is less affected by porin loss or active efflux resistance than many other β-lactam antibiotics. The aim of this study was to assess in vitro susceptibility to the cefiderocol of carbapenemase-producing gram-negative bacilli from clinical samples of hospitalized patients. Materials and methodsWe analyzed 102 clinical strains of carbapenemase-producing Enterobacterales and non-fermentives from hospital centers in Łódź, Poland. Antimicrobial susceptibility to cefiderocol was tested by the minimum inhibitory concentration test strips and disc diffusion methods. ResultsThe obtained results turned out to be ambiguous, and the area of technical uncertainty made their interpretation very difficult. ConclusionsThe cost of therapy with this antibiotic, and difficulties in interpreting the drug susceptibility are the limitations to the use of cefiderocol. Intensive work should be carried out to finally standardize an easily accessible and reliable method for the determination of susceptibility to cefiderocol.

Assessment of bacterial profile, antimicrobial susceptibility status, and associated factors of isolates among hospitalized patients at Dessie Comprehensive Specialized Hospital, Northeast Ethiopia

BackgroundAntimicrobial resistant bacteria among hospitalized patients are becoming a major public health threat worldwide, mainly in developing countries. Infections by these multidrug resistant pathogens cause high rate of mortality, prolong hospital stays, and affect individual and country economies in greater amounts. Thus, this study aimed to assess the bacterial profile, antimicrobial susceptibility status, and associated factors of isolates from hospitalized patients at the Dessie Comprehensive Specialized Hospital.MethodologyThis hospital-based cross-sectional study was conducted between February and April 2021. Consecutive sampling was used to select the study participants. All bacterial isolates were identified using standard bacteriological techniques. Antibiotic susceptibility testing was performed using disk diffusion technique. The data was analyzed using SPSS version 25. Descriptive statistics and logistic regression were used. A P-value of less than 0.05 was considered statistically significant.ResultsOf 384 clinical samples (blood, urine, stool, wound, vaginal discharge, and ear discharge) processed 180 (46.9%) were culture positive. Overall, Escherichia coli was the predominant isolate (41; 22.8%), followed by Staphylococcus aureus (36; 20%). Most of the isolates were from blood (70; 38.9%). The level of overall drug resistance of the gram-negative bacteria isolates for ampicillin, tetracycline, and cotrimoxazole was (104; 88.1%), (79; 75.9%), and (78; 75.0%), respectively. The overall multidrug rate of isolates was 143 (79.4%). Variables such as history of invasive procedures, chronic underlying diseases, history of hospitalization, and habit of eating raw animal products were statistically significant for the acquisition of bacterial infection.Conclusions and recommendationE. Coli and S. aureus were the most common isolates. Most of the isolates were resistant to commonly prescribed antibiotics. And also, consumption of raw animal products, chronic underlying disease, previous hospitalization, history of invasive procedures, and educational status were associated with the acquisition of bacterial infections. Therefore, routine antimicrobial susceptibility testing, proper patient management, wise use of antibiotics in clinical settings and health education are recommended.

Emergence of ceftazidime-avibactam resistance in blaKPC-33-harbouring ST11 Klebsiella pneumoniae in a paediatric patient

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially children. This study reports the infection caused by CRKP in a paediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that 12 strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC >64 mg/L) and restored the carbapenem susceptibility (Imipenem, MIC =0.25 mg/L; Meropenem, MIC =2 mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient Escherichia coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3–26 single nucleotide polymorphisms [SNPs]). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in paediatric-infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.

Antimicrobial Susceptibility Patterns of Enterococcus Species and Molecular Detection of Enterococcus faecalis Isolated from Patients with Urinary Tract Infection in a Tertiary Care Hospital in Bangladesh.

One of the most prevalent infectious diseases identified in both communities and hospitalized patientsis urinary tract infection (UTI). Enterococcus is evolved into a clinically pertinent uropathogen due to its evolving resistance to multiple antimicrobial agents.This study, detects antimicrobial susceptibility patterns of Enterococcus species and molecular detection of Enterococcus faecalis from patients with urinary tract infections. In this cross-sectional observational study, 165 urine samples were obtained from clinically diagnosed patients with UTIs of different ages and gender. Enterococcus species were identified by standard microbiological procedure and PCR (by using species-specific primers for Enterococcus faecalis). A modified Kirby Bauer Disc diffusion method was used to identify the antimicrobial susceptibility pattern following Clinical and Laboratory Standards Institute (CLSI) guidelines. Out of 165 urine samples, 134 samples yielded positive cultures. Enterococcus species were isolated from 23 (17.1%) urine samples. Among all Enterococcus, 16 (69.6%) isolates were E. faecalis, detected by PCR assay. A higher (30.4%) proportion of Enterococcus-positive patients were from the age group 48-57years and female patients (78.2%) had a higher prevalence. Enterococcal infection was found in 56.5% of non-catheterized patients and 43.5% of catheterized patients. Vancomycin and linezolid (78.3%) and meropenem (73.9%) sensitivity was prevalent among all Enterococcus species. They showed 100% resistance towards ceftriaxone, cefixime 95.7%, cefuroxime 91.3%, azithromycin 82.6%. This research indicated the occurrence of Enterococcus species and the advent of multidrug-resistant E. faecalis in patients with UTIs. Routine speciation and antimicrobial susceptibility testing of Enterococcus in various clinical samples is encouraged.

A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

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.

Prevalence, antimicrobial susceptibility profile, and associated risk factors of uropathogenic Escherichia coli among pregnant women attending Dr. Sumait Hospital Mogadishu, Somalia.

Uropathogenic Escherichia coli (UPEC) is a strain of E. coli commonly associated with urinary tract infections. In addition, antibiotic resistance in UPEC is one of the most significant health problems. This study was conducted to determine the prevalence, antimicrobial resistance, and factors linked to uropathogenic Escherichia coli (UPEC) in pregnant women. This cross-sectional study was conducted within a hospital setting between August 2022 and December 2022. Using consecutive convenient sampling, the research enrolled 220 pregnant women. The urine samples obtained from these women were cultured on MacConkey and blood agar and incubated at 37°C overnight, followed by sub-culturing on Mueller Hinton media. Bacterial identification involved Gram staining and biochemical characterization (TSI, indole, citrate, methyl red, urea agar, and motility tests). Conversely, susceptibility tests were performed using the Kirby-Bauer disk diffusion method. A binary logistic regression model and analysis of odds ratios (ORs) were employed to evaluate the risk factors associated with E. coli infection, and statistical significance was attributed to p-values of ≤0.05. Out of the 220 urine samples examined, 42 (19%) exhibited a positive culture, indicating an E. coli infection in pregnant women. Our analysis revealed that income, gestational age, and history of UTIs were identified as risk factors associated with E. coli infection. Most E. coli isolates demonstrated sensitivity to amikacin (100%), nitrofurantoin (85.7%), amoxicillin/clavulanic acid, and meropenem (83.3%). The prevalence of E. coli was remarkable. It could be recommended that pregnant women in antenatal care have routine culture and antimicrobial susceptibility tests to prevent transmission of resistant pathogens and complications in both pregnant mothers and the unborn baby.

Antimicrobial-Resistance Profile of Helicobacter pylori, Obtained from Endoscopic Patients in Bahir Dar, North West Ethiopia.

Antimicrobial resistance for Helicobacter pylori infection is a highly emerging problem throughout the world to treat gastric-associated diseases. People living in developing countries are more likely to acquire a Helicobacter pylori infection and less likely to gate treatment after infection due to poverty. Therefore, the current study was aimed at determining the magnitude and antibiotic-resistance profile of Helicobacter pylori isolated from patients who underwent endoscopic examination. A cross-sectional study was conducted from January to May 2019 at endoscopy service-providing health facilities that are found in Bahir Dar, Ethiopia (Gamby teaching general hospital, Kidane Mihret specialty higher clinic, and Eyasta specialty higher clinic). Data were collected using a pretested, structured questionnaire. Antibiotic susceptibility of Helicobacter pylori isolates from gastric biopsies was determined. Data were analyzed using SPSS version 23. The 17.8% proportion of Helicobacter pylori was isolated from 135 endoscopy-examined patients (24/135). The majority of isolates (71% of 17/24) were from males, while only 29% of 7/24) were from females. Antimicrobial-resistance of Helicobacter pylori was high to all commonly prescribed antibiotics: amoxicillin and metronidazole (91.7%), clarithromycin and ciprofloxacin (66.7% each), and tetracycline (37.5%). Helicobacter pylori isolates from the current study participants were rather low in number. But the highest antibiotic-resistance profile of Helicobacter pylori was observed. Therefore, these findings alarmingly indicate that routine antimicrobial susceptibility testing against Helicobacter pylori isolates is crucial for better patient management.

A Point Prevalence Survey of Antimicrobial Use at Geita Regional Referral Hospital in North-Western Tanzania.

We conducted a point prevalence survey (PPS) to determine the prevalence of antibiotic use at Geita Regional Referral Hospital (GRRH) located along the shores of Lake Victoria in north-western Tanzania. This has led to the identification of gaps for improvement. This PPS study was conducted on 9-10 March 2023. Patient-related information, including sociodemographic and clinical data, was collected from medical records. STATA software version 15.0 was used to perform descriptive data analysis. About 94.8% (55/58) patients were on antibiotics with a mean (±SD) prescription of 2 (±0.5) antibiotic agents ranging from 1 to 4 different agents. The commonest indications of the antibiotic prescription were medical prophylaxis 47.3% (26/55) followed by empiric treatment 41.8% (23/55). In total, 110 prescriptions were made, of which metronidazole (25.5%; n = 28), ceftriaxone (23.6%; n = 26), and ampicillin-cloxacillin (23.6%; n = 26) were frequently observed. Only 67.3% (n = 74) of prescriptions complied with Tanzania Standard Treatment Guidelines. Moreover, according to the WHO-AWaRe classification, 50.9%, 23.6%, and 25.5% were under the Access category, Watch category, and Not Recommended category, respectively. The prevalence of antibiotic use among patients admitted to GRRH was high, whereby medical prophylaxis and empiric treatment were the commonest indications for antibiotic prescription. To support rational therapy and antimicrobial stewardship initiatives, we recommend that laboratories in regional hospitals be equipped to conduct sustained routine culture and antimicrobial susceptibility testing.

Performance Evaluation of BD Phoenix and MicroScan WalkAway Plus for Determination of Fosfomycin Susceptibility in Enterobacterales.

Fosfomycin is an old bactericidal drug that has gained increasing interest in the last decade for its potential use in multi-drug resistant gram-negative infections. However, evidence on fosfomycin susceptibility testing reports a poor correlation between commercial methods vs. reference agar dilution (AD) for Enterobacterales (EB). The study aimed at assessing the performance of two automated systems for the determination of fosfomycin susceptibility in EB clinical isolates. Fosfomycin susceptibility testing results of two collections of 100 non-duplicate clinical EB strains obtained using two different platforms (BD Phoenix and MicroScan WalkAway Plus) were compared with those obtained by AD. Categorical agreement (CA), major error (ME) and very major error (VME) rates were calculated. BD Phoenix exhibited a 6.9% rate of false-resistant results and achieved a CA of 69%, whereas MicroScan WalkAway Plus achieved 3.7% of false-resistant results and 72% of CA. Both automated systems showed poor detection of resistant isolates, with 49.1% and 56.2% of false-susceptible results for BD Phoenix and Microscan WalkAway Plus, respectively. Overall, agar dilution remains the most suitable method for routine laboratory antimicrobial susceptibility testing of fosfomycin on Enterobacterales strains, given the poor performance of automated systems. The application of both automated systems, in the clinical laboratories reporting of fosfomycin, should be reviewed in light of the accuracy results falling below the acceptable threshold.

Emergence of high-level azithromycin-resistant Neisseria gonorrhoeae causing male urethritis in Johannesburg, South Africa, 2021.

In South Africa, Neisseria gonorrhoeae , which is the predominant cause of male urethritis, is treated syndromically using dual ceftriaxone and azithromycin therapy. We determined antimicrobial susceptibilities of N. gonorrhoeae isolates from urethral discharge specimens, and genetically characterised those with elevated minimum inhibitory concentrations (MICs) for first-line antimicrobials. Routine antimicrobial susceptibility testing (AST) of N. gonorrhoeae isolates included E-test for ceftriaxone, cefixime and gentamicin and agar dilution for azithromycin and spectinomycin. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) was performed for isolates with elevated MICs to identify antimicrobial resistance (AMR) determinants, and Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) was used to determine strain relatedness. N. gonorrhoeae was cultured from urethral discharge swab specimens obtained from 196 of 238 (82.4%) men presenting to a primary healthcare facility in Johannesburg in 2021. All viable isolates were susceptible to extended-spectrum cephalosporins. Four isolates had high azithromycin MICs ranging from 32mg/L to >256mg/L and grouped into two novel NG-MAST and NG-STAR groups. Two isolates from Group 1 (NG-MAST ST20366, NG-STAR ST4322) contained mutated mtrR (G45D) and 23S rRNA (A2059G) alleles, while the two isolates from Group 2 (NG-MAST ST20367, NG-STAR ST4323) had different mutations in mtrR (A39T) and 23S rRNA (C2611T). We report the first cases of high-level azithromycin resistance in N. gonorrhoeae from South Africa. Continued AMR surveillance is critical to detect increasing azithromycin resistance prevalence in N. gonorrhoeae , which may justify future modifications to the STI syndromic management guidelines.

Clinically undetected polyclonal heteroresistance among Pseudomonas aeruginosa isolated from cystic fibrosis respiratory specimens.

Pseudomonas aeruginosa infection is the leading cause of death among patients with cystic fibrosis (CF) and a common cause of difficult-to-treat hospital-acquired infections. P. aeruginosa uses several mechanisms to resist different antibiotic classes and an individual CF patient can harbour multiple resistance phenotypes. To determine the rates and distribution of polyclonal heteroresistance (PHR) in P. aeruginosa by random, prospective evaluation of respiratory cultures from CF patients at a large referral centre over a 1 year period. We obtained 28 unique sputum samples from 19 CF patients and took multiple isolates from each, even when morphologically similar, yielding 280 unique isolates. We performed antimicrobial susceptibility testing (AST) on all isolates and calculated PHR on the basis of variability in AST in a given sample. We then performed whole-genome sequencing on 134 isolates and used a machine-learning association model to interrogate phenotypic PHR from genomic data. PHR was identified in most sampled patients (n = 15/19; 79%). Importantly, resistant phenotypes were not detected by routine AST in 26% of patients (n = 5/19). The machine-learning model, using the extended sampling, identified at least one genetic variant associated with phenotypic resistance in 94.3% of isolates (n = 1392/1476). PHR is common among P. aeruginosa in the CF lung. While traditional microbiological methods often fail to detect resistant subpopulations, extended sampling of isolates and conventional AST identified PHR in most patients. A machine-learning tool successfully identified at least one resistance variant in almost all resistant isolates by leveraging this extended sampling and conventional AST.

IN VITRO ACTIVITY OF CEFTAZIDIME-AVIBACTAM AND COLISTIN AGAINST CARBAPENEM-RESISTANT PSEUDOMONAS AERUGINOSA CLINICAL ISOLATES

Objective: Infections caused by multi drug-resistant Gram-negative bacilli are increasingly reported worldwide. Ceftazidime-avibactam is a novel antibiotic combination that presents good activity against carbapenem-resistant Enterobacterales members and Pseudomonas aeruginosa isolates. The objective of this study was to evaluate the in vitro activity of ceftazidime-avibactam and colistin against carbapenem-resistant P. aeruginosa isolates. Materials and Methods: A total of 100 carbapenem-resistant and non-duplicate P. aeruginosa isolates obtained from patient samples in our hospital between 2016-2021 were included in the study. The isolates were identified by MALDI-TOF MS (Bruker Daltonics, Germany). The minimum inhibitory concentration (MIC) values of meropenem, colistin, ceftazidime, and ceftazidime-avibactam were determined by the broth microdilution method. The presence of carbapenemase genes bla KPC, bla NDM, bla OXA-48, bla IMP, and bla VIM were investigated using PCR. Results: Carbapenemase genes were not detected among the isolates except for one isolate producing bla VIM. The susceptibility rates of ceftazidime-avibactam and colistin were 90% (n=90) and 100% (n=100), respectively. The MIC50 and MIC90 values for meropenem, ceftazidime, ceftazidime-avibactam, and colistin against P. aeruginosa isolates were found to be 32/64, 8/64, 4/8, 0.5/2 µg/mL, respectively.&nbsp;Conclusion: The data suggests that ceftazidime-avibactam exhibits as a viable alternative for the treatment of carbapenem-resistant P. aeruginosa isolates. It’s noteworthy that colistin resistance was not detected among the study isolates. Rational use of antibiotics should be emphasized to prevent the development of antibiotic resistance. Surveillance of ceftazidime-avibactam and colistin should be followed up with routine antimicrobial susceptibility tests.

Diarrhea-Causing Bacteria and Their Antibiotic Resistance Patterns Among Diarrhea Patients From Ghana.

Diarrheal disease remains a major global health problem particularly in children under 5 years and the emergence of antibiotic-resistant strains of causative pathogens could slow control efforts, particularly in settings where treatment options are limited. This surveillance study conducted in Ghana aimed to determine the prevalence and antimicrobial susceptibility profile of diarrhea-causing bacteria. This was a cross-sectional study carried out in five health facilities in the Ga West Municipality of Ghana between 2017 and 2021. Diarrheic stool samples from patients were collected and cultured on standard differential/selective media and isolates identified by standard biochemical tests, MALDI-TOF assay, and serological analysis. The antibiogram was determined using Kirby-Bauer disk diffusion and Microscan autoScan4 MIC panels which were used for extended-spectrum beta-lactamase (ESBL) detection. Bacteria were isolated from 97.5% (772/792) of stool samples, and 167 of the isolates were diarrheagenic and met our inclusion criteria for antimicrobial resistance (AMR) analysis. These included Escherichia coli (49.1%, 82/167), Salmonella species (23.9%, 40/167), Vibrio species (16.8%, 28/167), and Shigella species (10.2%, 17/167). Among 24 Vibrio species, we observed resistances to cefotaxime (21/24, 87.5%), ceftriaxone (20/24, 83.3%), and ciprofloxacin (6/24, 25%), including four multi-drug resistant isolates. All 13 Vibrio parahaemolyticus isolates were resistant to cefazolin. All 17 Shigella isolates were resistant to tetracycline with resistance to shigellosis drugs such as norfloxacin and ciprofloxacin. Salmonella isolates were highly susceptible to norfloxacin (40/40, 100%) and tetracycline (12/34, 35%). Two ESBL-producing E. coli were also identified with marked susceptibility to gentamicin (66/72, 91.7%) and amikacin (57/72, 79.2%) prescribed in the treatment of E. coli infections. This study showed the different bacteria implicated in diarrhea cases in Ghana and the need for differential diagnoses for better treatment outcomes. Escherichia coli, Shigella, Salmonella, and Vibrio have all been implicated in diarrhea cases in Ghana. The highest prevalence was E. coli and Salmonella with Shigella the least prevalent. Resistance to commonly used drugs found in these isolates may render bacteria infection treatment in the near future nearly impossible. Routine antimicrobial susceptibility testing, effective monitoring, and nationwide surveillance of AMR pathogens should be implemented to curb the increase of antimicrobial resistance in Ghana.

Discrimination of Methicillin-resistant Staphylococcus aureus by MALDI-TOF Mass Spectrometry with Machine Learning Techniques in Patients with Staphylococcus aureus Bacteremia.

Early administration of proper antibiotics is considered to improve the clinical outcomes of Staphylococcus aureus bacteremia (SAB), but routine clinical antimicrobial susceptibility testing takes an additional 24 h after species identification. Recent studies elucidated matrix-assisted laser desorption/ionization time-of-flight mass spectra to discriminate methicillin-resistant strains (MRSA) or even incorporated with machine learning (ML) techniques. However, no universally applicable mass peaks were revealed, which means that the discrimination model might need to be established or calibrated by local strains’ data. Here, a clinically feasible workflow was provided. We collected mass spectra from SAB patients over an 8-month duration and preprocessed by binning with reference peaks. Machine learning models were trained and tested by samples independently of the first six months and the following two months, respectively. The ML models were optimized by genetic algorithm (GA). The accuracy, sensitivity, specificity, and AUC of the independent testing of the best model, i.e., SVM, under the optimal parameters were 87%, 75%, 95%, and 87%, respectively. In summary, almost all resistant results were truly resistant, implying that physicians might escalate antibiotics for MRSA 24 h earlier. This report presents an attainable method for clinical laboratories to build an MRSA model and boost the performance using their local data.