ACTA Microbiologica Hellenica (Volume 68, Issue 2)

Determining antimicrobial susceptibility and antimicrobial resistance mechanisms in the clinical laboratory: where are we and where are we heading?

Nektaria Rekleiti
Laboratory of Microbiology, Tzaneio General Hospital of Piraeus

Since the discovery of penicillin, almost a century ago, the battle against resistant microbes has been fierce but uneven. Microbes have been proven quite adaptable and have developed many different antimicrobial resistance (AMR) mechanisms to evade antibiotics. These AMR mechanisms mainly regulate cephalosporin and carbapenem resistance for Gram-negative bacteria, methicillin resistance for Staphylococcus aureus and vancomycin resistance for Enterococci. Greece is endemic for most known multi-drug resistant organisms (MDRO) that often cause community and hospital-acquired infections, perplexing treatment, increasing length of stay in the hospital and relevant costs, and increasing morbidity and mortality. Detection and treatment of such infections in a timely and effective way is imperative. For the past few decades, scientists from different scientific fields have been developing technologies and methods to assist the early and reliable detection of AMR to optimize not only treatment but also infection control practices in an effort to restrain it. This review focuses on current practices to detect AMR and the corresponding resistance mechanisms. From the well-established classic diffusion antibiogram to the rapid automated tools that provide susceptibility profiles of bacteria within a few hours, and from the time-consuming phenotypic AMR detection methods to rapid molecular AMR mechanism detection directly from the sample, Microbiology has come a long way. Most microbiological laboratories are currently using a combination of phenotypic and molecular methods for AMR detection, in an effort to make the best out of both. Integrating novel technologies into the laboratory routine workflow has its challenges, with the financial burden being one of the most significant. However, if the progress in the field of Microbiology since the emergence of the first resistant microbe until now is any indication, the future holds many more adventures and scientific breakthroughs in the fight against AMR.

Antimicrobial susceptibility; antimicrobial resistance mechanisms; phenotypic resistance detection; molecular resistance detection

Investigation of virulence factors and their relationship with antimicrobial resistance among uropathogenic Escherichia coli isolates identified from patients in Basrah city, Iraq.

Huda Al-Moslem 1; Seyedeh Elham Rezatofighi 1; Yasin Yacoup Yousif AL-Luaibi 2;
Mohammad Reza Akhoond 3
1 Department of Biology, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
2 Department of Biology, College of Science; University of Basrah; Basrah; Iraq
3 Mathematical Sciences and Computer Faculty, Shahid Chamran University of Ahvaz, Ahvaz, Iran

Uropathogenic Escherichia coli (UPEC) is one of the main causes of urinary tract infections (UTIs). We aimed to investigate the antimicrobial resistance (AMR) pattern, the frequency of some virulence genes (VGs), and the association of AMR with VGs. A total of 300 urine samples were collected from patients suspected to have UTI. The samples were examined by biochemical and microbiological methods and VITEK2 compact system to identify the bacterial infectious agents. The antimicrobial resistance pattern and virulence genes (papAH, papC, papEF, papG, fimH, and fyuA) profile of UPEC isolates were investigated and the relationship between these traits was evaluated by statistical methods.
Among these samples, 201 (67%) exhibited a positive growth on culture media. E. coli was isolated from 60 (29.85 %) specimens followed by Klebsiella pneumoniae 42 (20.90%), Staphylococcus aureus 38 (18.9%), Enterobacter spp 29 (14.43%), Pseudomonas aeruginosa 10 (4.98%), Proteus mirabilis 15 (7.46%), others about 7 (3.48 %) isolates. Antibiogram results of 15 antibiotics examined showed that all E. coli isolates were multidrug-resistant (MDR). The commonest antimicrobial resistance was observed against Streptomycin (100%), Kanamycin (98.3%), and Ampicillin (96.7%). The most sensitive agents were Meropenem (96.4%), Nitrofurantoin (93.4%), and Imipenem (85%). VGs detected among UPEC isolates were fimH (88.3%), papAH (85%), papC (85%), papG (80%), fyuA (80%), and papEF (60%).These results alleged no strong correlation between VGs and AMR in E. coli strains. Based on the results of the present study, virulence genes, and antimicrobial resistance are independent properties and can transfer to other bacteria separately.Further studies are needed to better understand the relationship between different virulence factors (VFs) and AMR at a molecular level, as most UPEC isolates express several VFs and AMR simultaneously.

Uropathogenic Escherichia coli; Antimicrobial Resistance; Virulence genes; Urinary tract infection

Differences in the detection of virulence genes among clinical isolates of Coagulase-Negative Staphylococci

Anastasios Vogiatzakis 1, Despina Tassi-Papatheou 2, Lemonia Skoura 3, Konstantinos Tsiamis 4,
Athanassios Tsakris 1, Georgia Vrioni 1, Joseph Papaparaskevas 1,2
1 Department of Microbiology, Medical School, National & Kapodistrian University of Athens, 11527, Athens, Greece.
2 Central Diagnostic Laboratories, Onassis Cardiac Surgery Center, 17674, Kallithea, Greece.
3 Department of Microbiology, AHEPA University Hospital, Medical School, Aristotle University of Thessaloniki, 54636, Thessaloniki, Greece.
4 Department of Public and One health, University of Thessaly, 43100, Karditsa, Greece.

Although Coagulase Negative Staphylococci (CoNS) have been considered for years as colonizers of the human body, it is now clear that they can cause serious infections, especially among immunocompromised patients. Various genes express for pathogenic mechanisms, including the icaA and icaD which enhance biofilm production, as well as genes than encode for staphylococcal enterotoxins (sea, seb) and the toxic shock syndrome toxin (tst). The aim of this study was to detect and determine through PCR the presence and distribution of these genes in CoNS strains isolated from clinical specimens. The results of the study showed that icaA and icaD genes were present in 49% and 52% of CoNS strains respectively, while 36,5% of the strains harbored the tst gene. No CoNS strains harbored the enterotoxin A and B genes. The S. epidermidis strains harbored the icaA/D and tst genes more frequently than the remaining CoNS species (statistically significant difference, p<0.001 and p=0.001, respectively). Keywords:
Coagulase Negative Staphylococci (CoNS); virulence genes; enterotoxins; the toxic shock syndrome toxin.

Workflow for microbiological diagnosis of bacterial gastroenteritis combining a molecular assay as first-line with reflective stool culture

Maria Jose Munoz-Davila 1,2, C. Candel-Pérez 1, M.R. Vicente 1, M. Piqueras 2, J.M. Artero 1
1 Microbiology laboratory. General University Hospital Reina Sofia, Murcia. Spain.
2 Microbiology and Genetics Department, Faculty of Medicine, University of Murcia, Murcia Spain.

Conventional microbiological methods for bacterial enteric disease diagnosis are time-consuming, labour intensive and provide low sensitivity. The aim of this study was to evaluate the results of a new diagnosis strategy which replaces traditional stool culture with a molecular detection using the BD MAX™ System (BD Life Sciences, Sparks, Maryland, United States) as first-line assay together with reflective culture. A total of 1.590 specimens were prospectively requested for stool culture. The molecular detection included the BD MAX enteric bacterial panel together with the BD MAX extended enteric bacterial panel (BDM GIP) performed simultaneously on the same stool specimen. In 18.8% of specimens (176 of the 936 valid samples) there was one or more than one target positive with the following percent positivity: 9.7% Campylobacter spp., 5.7% Salmonella spp., 1.3% Shiga toxin genes (stx1/stx2), 1.2% Shigella spp./enteroinvasive Escherichia coli (EIEC), 1% Yersinia enterocolitica, 1% Vibrio spp. (V. vulnificus/V. parahaemolyticus/V. cholerae), 0.3% Plesiomonas shigelloides, and 0.2% Enterotoxigenic E. coli (ETEC) enterotoxin LT/ST genes. Positive reflective stool culture noted a correlation of 69.5% with the molecular test, missing 23.9% and 15.4% in the cases of Campylobacter spp., and Salmonella spp., respectively. In conclusion, this clinical study demonstrated very good performance of the BDM GIP. The performance and ease of use may provide advantages to many laboratories, improving the detection of bacterial stool pathogens and time to reporting results.

bacterial gastroenteritis, molecular detection, BD MAX™ System.

Diagnostic performance of the serology screening testing of blood donors. Retrospective study (2018–2022) conducted at the Serology Laboratory of AHEPA Blood Center

Magdalini Pape, Vasiliki Bakaloudi, Parthena Lazaridou, Dimitris, Pisokas, Pashalia Poulioudi, George Nikolaidis, Aikaterini Boukouvala, Christina Andrikopoulou, Soultana Nikolaidou, Iliana Pentsiou, Chariklia Pafili, Maria Chatzikyrkou
Serology Laboratory, Blood Center, AHEPA University General Hospital of Thessaloniki

The use of screening tests in a blood donor population, a population with a low prevalence of infectious markers, is a complex process. All assays should have high levels both of sensitivity and specificity. Increased sensitivity is of outmost importance to detect and remove potentially infectious blood products from the blood supply and eliminate false negative results. At the same time, specificity is also important in reducing the number of false positive results, in order to avoid unnecessary exclusion of certain donors. The aim of our study was to analyze the seroprevalence of transfusion-transmissible infectious agents of voluntary blood donors within the period 2018–2022 and verify the accuracy of screening tests followed by confirmatory assays. The study conducted at the Serology Laboratory of AHEPA Blood Center. Blood samples were tested for syphilis, HBsAg, anti-HCV, anti-HTLV I/II and HIV Ag/Ab. The screening strategy included repeated testing of reactive samples, followed by confirmatory testing. Our results confirmed the low prevalence of transfusion transmitted infections in Greek blood donors. As a consequence, the positive predictive value of the screening assays was poor, something not attributed to poor performance of the selected screening tests, and a relevant number of uninfected donations were excluded. Specificity for all assays was within our expectations and definitely equal or better than the value demonstrated on the reagent inserts. The use of the confirmatory algorithm offered valuable help in the management of blood donors.

blood donor population; algorithm; diagnostic accuracy; specificity.