- Volume 70, Issue 3, 2021

2020 was the year when microbiology burst onto the world stage, not just as the science of small living things, but as the prism through which we understood global events. Clinical logic suffered under pressure arising from an urgent need to confirm or exclude severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This is a generation’s Hobbesian moment in which the public concern for safety and security from infection outweighs the pursuit of personal freedom. The strangeness of a world in which a minute particle wields superhuman power has generated its list of unlikely heroes and mendacious villains. As the year comes to an end, there are glimmers of light amid the gloom: the prospect of an effective vaccine, and life after the pandemic.

In recent decades there has been an increase in knowledge of the distribution, species diversity and growth patterns of bacteria in human chronic infections. This has challenged standard diagnostic methods, which have undergone a development to both increase the accuracy of testing as well as to decrease the occurrence of contamination. In particular, the introduction of new technologies based on molecular techniques into the clinical diagnostic process has increased detection and identification of infectious pathogens. Sampling is the first step in the diagnostic process, making it crucial for obtaining a successful outcome. However, sampling methods have not developed at the same speed as molecular identification. The heterogeneous distribution and potentially small number of pathogenic bacterial cells in chronic infected tissue makes sampling a complicated task, and samples must be collected judiciously and handled with care. Clinical sampling is a step in the diagnostic process that may benefit from innovative methods based on current knowledge of bacteria present in chronic infections. In the present review, we describe and discuss different aspects that complicate sampling of chronic infections. The purpose is to survey representative scientific work investigating the presence and distribution of bacteria in chronic infections in relation to various clinical sampling methods.

Over a decade ago, a multidrug-resistant nosocomial fungus Candida auris emerged worldwide and has since become a significant challenge for clinicians and microbiologists across the globe. A resilient pathogen, C. auris survives harsh disinfectants, desiccation and high-saline environments. It readily colonizes the inanimate environment, susceptible patients and causes invasive infections that exact a high toll. Prone to misidentification by conventional microbiology techniques, C. auris rapidly acquires multiple genetic determinants that confer multidrug resistance. Whole-genome sequencing has identified four distinct clades of C. auris, and possibly a fifth one, in circulation. Even as our understanding of this formidable pathogen grows, the nearly simultaneous emergence of its distinct clades in different parts of the world, followed by their rapid global spread, remains largely unexplained. We contend that certain host–pathogen–environmental factors have been evolving along adverse trajectories for the last few decades, especially in regions where C. auris originally appeared, until these factors possibly reached a tipping point to compel the evolution, emergence and spread of C. auris. Comparative genomics has helped identify several resistance mechanisms in C. auris that are analogous to those seen in other Candida species, but they fail to fully explain how high-level resistance rapidly develops in this yeast. A better understanding of these unresolved aspects is essential not only for the effective management of C. auris patients, hospital outbreaks and its global spread but also for forecasting and tackling novel resistant pathogens that might emerge in the future. In this review, we discuss the emergence, spread and resistance of C. auris, and propose future investigations to tackle this resilient pathogen.

Dermatophytosis is a common cutaneous mycosis worldwide whose prevalence in Brazil is still unknown. This systematic review has estimated the burden of dermatophytoses from updated literature data reported in the general Brazilian population. We used the following databases: Web of Science, Medline/PubMed, Embase, The Cochrane Library and Scopus for studies published between 2011 and 2020. Original articles with an emphasis on prevalence data for dermatophytosis in the Brazilian population, and diagnosed by culture exam or molecular biology were eligible. We also assessed the methodological quality of the studies. A total of 24 articles met the inclusion criteria and were reviewed. The occurrence of dermatophytoses found in the studies ranged from 4–88.50 %. The pooled prevalence of dermatophytosis for the population studies was 25 % (95 % CI: 24.7–25.3 %). The size of the samples used in the studies ranged from 45 to 36 446 participants, and ages ranged up to 98 years old. The populations studied involved mostly women. The presence of tinea unguium (toenail and fingernail) and tinea pedis were the most frequent dermatophytosis, and we observed a predominance of Trichophyton rubrum, T. interdigitale and T. mentagrophytes. The studies were primarily conducted in patient groups with suspected mycoses and were not entirely representative of the general population. Yet we believe that in the future, more collaborative strategies would improve both diagnostic capacity and epidemiological methodologies, associating the prevalence of dermatophytosis with social and environmental risk factors. This review helps to better understand future epidemiological trends in Brazil and the world.

Bacteria of the genus Streptococcus , earlier considered typically animal, currently have also been causing infections in humans. It is necessary to make clinicians aware of the emergence of new species that may cause the development of human diseases. There is an increasing frequency of isolation of streptococci such as S. suis , S. dysgalactiae , S. iniae and S. equi from people. Isolation of Streptococcus bovis/Streptococcus equinus complex bacteria has also been reported. The streptococcal species described in this review are gaining new properties and virulence factors by which they can thrive in new environments. It shows the potential of these bacteria to changes in the genome and the settlement of new hosts. Information is presented on clinical cases that concern streptococcus species belonging to the groups Bovis, Pyogenic and Suis. We also present the antibiotic resistance profiles of these bacteria. The emerging resistance to β-lactams has been reported. In this review, the classification, clinical characteristics and antibiotic resistance of groups and species of streptococci considered as animal pathogens are summarized.

Klebsiella pneumoniae causes a diversity of infections in both healthcare and community settings. This pathogen is showing an increased ability to accumulate antimicrobial resistance and virulence genes, making it a public health concern. Here we describe the whole-genome sequence characteristics of an ST15 colistin-resistant K. pneumoniae isolate obtained from a blood culture of a 79-year-old female patient admitted to a university hospital in Brazil. Kp14U04 was resistant to most clinically useful antimicrobial agents, remaining susceptible only to aminoglycosides and fosfomycin. The colistin resistance in this isolate was due to a ~1.3 kb deletion containing four genes, namely mgrB, yebO, yobH and the transcriptional regulator kdgR. The study isolate presented a variety of antimicrobial resistance genes, including the carbapenemase-encoding gene bla KPC-2, the extended-spectrum beta-lactamase (ESBL)-encoding gene bla SHV-28 and the beta-lactamase-encoding gene bla OXA-1. Additionally, Kp14U04 harboured a multiple stress resistance protein, efflux systems and regulators, heavy metal resistance and virulence genes, plasmids, prophage-related sequences and genomic islands. These features revealed the high potential of this isolate to resist antimicrobial therapy, survive in adverse environments, cause infections and overcome host defence mechanisms.

Klebsiella pneumoniae strains carrying OXA-48-like carbapenemases are increasingly prevalent across the globe. There is thus an urgent need to better understand the mechanisms that underpin the dissemination of bla OXA-48-like carbapenemases. To this end, four ertapenem-resistant K. pneumoniae isolates producing OXA-48-like carbapenemases were isolated from two patients. Genome sequencing revealed that one sequence type (ST) 17 isolate carried bla OXA-181, whilst three isolates from a single patient, two ST76 and one ST15, carried bla OXA-232. The 50514 bp bla OXA-181-harbouring plasmid, pOXA-181_YML0508, was X3-type with a conjugation frequency to Escherichia coli of 1.94×10−4 transconjugants per donor. The bla OXA-232 gene was located on a 6141 bp ColKP3-type plasmid, pOXA-232_WSD, that was identical in the ST76 and ST15 K. pneumoniae isolates. This plasmid could be transferred from K. pneumoniae to E. coli at low frequency, 8.13×10−6 transconjugants per donor. Comparative analysis revealed that the X3 plasmid acquired the bla OXA-48-like gene via IS3000-mediated co-integration of the ColKP3-type plasmid. Our study highlights how plasmid integration and rearrangements can contribute to the spread of bla OXA-48-like genes, which provides important clues for clinical prevention of the dissemination of K. pneumoniae strains carrying bla OXA-48-like carbapenemases.

The genus Candida spp. has been highlighted as one of the main etiological agents causing fungal infections, with Candida albicans being the most prominent, responsible for most cases of candidemia. Due to its capacity for invasion and tissue adhesion, it is associated with the formation of biofilms, mainly in the environment and hospital devices, decreasing the effectiveness of available treatments. The repositioning of drugs, which is characterized by the use of drugs already on the market for other purposes, together with molecular-docking methods can be used aiming at the faster development of new antifungals to combat micro-organisms. This study aimed to evaluate the antifungal effect of diazepam on mature C. albicans biofilms in vitro and its action on biofilm in formation, as well as its mechanism of action and interaction with structures related to the adhesion of C. albicans, ALS3 and SAP5. To determine the MIC, the broth microdilution test was used according to protocol M27-A3 (CLSI, 2008). In vitro biofilm formation tests were performed using 96-well plates, followed by molecular-docking protocols to analyse the binding agent interaction with ALS3 and SAP5 targets. The results indicate that diazepam has antimicrobial activity against planktonic cells of Candida spp. and C. albicans biofilms, interacting with important virulence factors related to biofilm formation (ALS3 and SAP5). In addition, treatment with diazepam triggered a series of events in C. albicans cells, such as loss of membrane integrity, mitochondrial depolarization and increased production of EROs, causing DNA damage and consequent cell apoptosis.

Introduction. Resistance to rifampin (RIF) in Mycobacterium tuberculosis infection is associated with mutations in the rpoB gene coding for the β-subunit of RNA polymerase. The contribution of various rpoB mutations to the development and level of RIF resistance remains elusive.

Hypothesis/Gap Statement. Various rpoB mutations may be associated with differential levels of RIF resistance.

Aim. This study aimed to investigate the relationship between specific rpoB mutations and the MICs of RIF and rifabutin (RFB) against M. tuberculosis .

Methodology. Of the 195 clinical isolates, 105 and 90 isolates were randomly selected from isolates resistant to RIF and sensitive to RIF, respectively. The MICs of 12 agents for M. tuberculosis isolates were determined using commercial Sensititre M. tuberculosis MIC plates and the broth microdilution method. Strains were screened for rpoB mutations by DNA extraction, rpoB gene amplification and DNA sequence analysis.

Results. One hundred isolates (95.24 %) were found to have mutations in the RIF-resistance-determining region (RRDR) of the rpoB gene. Three rpoB mutations were identified in 90 RIF-susceptible isolates. Out of 105 isolates, 86 (81.90 %) were cross-resistant to both RIF and RFB. The most frequent mutation occurred at codons 450 and 445. We also found a novel nine-nucleotide (ATCATGCAT) deletion (between positions 1543 and 1551) in the rpoB gene in two strains (1.90 %) with resistance to RIF, but susceptibility to RFB. In addition, the mutation frequency at codon 450 was significantly higher in RIF-resistant/RFB-resistant (RIFR/RFBR) strains than in RIFR/RFBS strains (75.58 % versus 21.05 %, P<0.01), whereas the mutation frequency at codon 435 was significantly lower in RIFR/RFBR strains than in RIFR/RFBS strains (1.16 % versus 26.32 %, P<0.01).

Conclusion. Our data support previous findings, which reported that various rpoB mutations are associated with differential levels of RIF resistance. The specific mutations in the rpoB gene in RIFR/RFBR isolates differed from those in the RIFR/RFBS isolates. A novel deletion mutation in the RRDR might be associated with resistance to RIF, but not to RFB. Further clinical studies are required to investigate the efficacy of RFB in the treatment of infections caused by M. tuberculosis strains harbouring these mutations.

Introduction. Since mcr-1 was first reported in China, there have been ten variants of MCR appearing nationwide so far. Multidrug-resistant Enterobacteriaceae bacteria carrying both NDM and MCR have become a serious threat to global public health.

Hypothesis/Gap Statement. The genetic structure of mcr-9 needs to be better understood in order to better prevent and control the transmission of drug-resistant genes.

Aims. The aim of this study was to characterize the presence of two Enterobacter hormaechei isolates, which carries bla NDM-5 CME2 and the coexistence of mcr-9 and bla NDM-1 strain CMD2, which were isolated from a patient with diabetes in Sichuan, China.

Methodology. The microbroth dilution method was used for antibiotic susceptibility. Conjugation experiment was used to investigate the transferability of bla NDM-1, bla NDM-5 and mcr-9. Whole-genome sequencing was performed on Illumina HiSeq platform. The ability of biofilm formation was detected by crystal-violet staining, the virulence of the bacteria was measured by Galleria mellonella killing assay.

Results. bla NDM-5 carrier CME2 and CMD2 with bla NDM-1 and mcr-9 were resistant to carbapenems, β-lactam, aminoglycoside, quinolone and tetracycline, while CMD2 was also resistant to colistin. Conjugation assay and plasmid replicon typing further demonstrated that both bla NDM-1 and bla NDM-5 were respectively present on the self-transferrable IncX3 plasmid, mcr-9 was located on the self-transferrable IncHI2 plasmid. Through the analysis of mcr-9 gene context, the structure was DUF4942-rcnR-rcnA-copS-IS903-mcr-9-wbuC-qseC-qseB-IS1R-ΔsilR-IS903, bla NDM-1 context was IS3000-ΔISAba125-IS5-bla NDM-1-ble-trpF-groS-groL-insE-ΔIS26 structure, bla NDM-5 structure was IS3000-bla NDM-5-ble-trpF-dsbC-ΔIS26-umuD-ISKox3-tnpR-parA. Biofilm formation of CME2 was stronger than CMD2. There was no significant difference in virulence between the two strains.

Conclusion. This study reveals two multiple drug-resistant E. hormaechei isolates from diabetes patient samples. E. hormaechei carrying two NDM-resistant genes is already a serious threat, where MCR is an important cause of treatment failure in bacterial infections. This study is a reminder not only to prevent infection in patients with diabetes, but also to constantly monitor the epidemic and spread of the drug-resistant gene.

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance–nodulation–cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1–carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1–carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

This study aimed to evaluate whether the antibiotic fidaxomicin has in vitro activity against Mycobacterium tuberculosis (Mtb). 38 fully drug-sensitive Mtb strains and 34 multidrug-resistant tuberculosis (MDR-TB) strains were tested using the microplate alamar blue assay (MABA) method to determine the minimum inhibitory concentrations (MICs) for fidaxomicin and rifampicin. Fidaxomicin has high in vitro activity against Mtb and is a potential drug to treat Mtb, and MDR-TB infections in particular.

Introduction. Mycobacterium abscessus complex (MABC) is an infectious agent associated with macrolide resistance and treatment failure.

Hypothesis/Gap Statement. Despite drug-susceptibility testing for MABC isolates including clarithromycin (CAM), long-term treatment with azithromycin (AZM) for MABC disease is recommended.

Aim. We compared phenotypic and genotypic resistance to AZM and CAM in clinical isolates and evaluated the accumulation of intrinsic macrolide resistance (AIM) and morphological changes by macrolides exposure.

Methodology. Forty-nine isolates were characterized regarding erm(41) sequevars. Sequencing data were compared to the nucleotide sequence of rrl and whiB7. The AIM MIC was performed in three reference strains and 15 isolates were randomized [each set of five isolates with M. abscessus subsp. abscessus (MAA) T28, MAA C28 and subsp. massiliense (MAM)].

Results. The 49 isolates were distributed as 24 MAA T28, 5 MAA C28 and 20 MAM. The MIC50 values to CAM at day 3 in MAA T28, C28 and MAM were 1, 0.12 and 0.12 µg ml−1, while those at day 14 were 32, 0.5 and 0.12 µg ml−1, respectively. The AZM-MIC50 values at day 3 of the above isolates were 4, 0.25 and 0.5 µg ml−1, while those at day 14 were >64, 0.5 and 0.5 µg ml−1, respectively. Neither mutations in rrl of MAA T28 with acquired resistance nor deletions in whiB7 of MAA T28 without inducible resistance were observed . For AIM MIC, MAA T28 showed that the time-to-detection of AZM resistance was significantly faster over that of CAM (P<0.05). Morphological changes were not determined in all isolates.

Conclusion. Our findings did not support the suggestion for the preferential use of AZM for, at least, MAA T28 disease due to the high-level MIC value and the increased AIM. The long duration of AZM-based treatment eventually may favour the emergence of isolates with a high-level of intrinsic resistance.

Introduction. The possible transfer of antimicrobial resistance genes between Enterococcus faecium isolates from humans and different animal species, including those not covered by monitoring programs (e.g. pet and wildlife), poses a serious threat to public health.

Hypothesis/Gap Statement. Little is known about occurrence and mechanisms of phenomenon of multidrug resistance of E. faecium isolated from various host species in Poland.

Aim. The aim of the study was to characterize multidrug-resistant E. faecium isolated from humans and animals (livestock, pets and wildlife) in terms of the occurrence of genetic markers determining resistance.

Methodology. Bacterial isolates were tested for phenotypic resistance and the presence of genes encoding resistance to macrolides, tetracycline, aminoglycosides, aminocyclitols and phenicols as well as efflux pump (emeA), resolvase (tndX) and integrase (Int-Tn) genes. The quinolone resistance-determining regions of gyrA and parC were sequenced.

Results. Human isolates of E. faecium were characterized by high-level resistance to: ciprofloxacin, enrofloxacin, erythromycin (100 %), as well, as aminoglycosides resistance (kanamycin – 100%, streptomycin – 78 %, gentamicin – 78%). Regardless of the animal species, high level of resistance of E. faecium to tetracycline (from 88–100 %), erythromycin (from 82–94 %) and kanamycin (from 36–100 %) was observed. All E. faecium isolates from wildlife were resistant to fluoroquinolones. However, full susceptibility to vancomycin was observed in all isolates tested. Phenotypic antimicrobial resistance of E. faecium was identified in the presence of the following resistance genes: erm(B) (70%), msr(A) (50 %), tet(L) (35 %), tet(K) (34 %), tet(M) (76 %), aac(6’)-Ie-aph(2″)-Ia (25%), ant(6)-Ia (31%), aph(3)-IIIa (68 %), (tndX) (23 %), and integrase gene (Int-Tn) (34 %). A correlation between an amino acid substitution at positions 83 and 87 of gyrA and position 80 of parC and the high-level fluoroquinolone resistance in E. faecium has been observed as well.

Conclusion. The level and range of antimicrobial resistance and the panel of resistance determinants is comparable between E. faecium isolates, despite host species.

Introduction. Blood culture (BC) remains the gold standard for the diagnosis of bloodstream infection. Clinical microbiology laboratories must ensure the quality of their BC process from receipt to definitive results.

Aim. In this study, we followed the evolution of different quality indicators for BCs over the first year of implementation of the BacT/Alert Virtuo system in a French hospital.

Methodology. In our laboratory, we instituted regular monitoring of several quality indicators to track (i) delays in sample registration, (ii) delays in loading BC bottles in our incubating system (BacT/Alert Virtuo) after registration, (iii) the volume of blood in bottles and (iv) the contamination rates.

Results. For 53 892 BC bottles loaded in the BacT/Alert Virtuo from 23 January to 31 December 2019, the delays in sample registration, loading and unloading were respectively 3.5 h±0.016, 44 min±0.209 and 5.8 h±0.0727. Intriguingly, the automated process performed by the BacT/Alert Virtuo system to check the blood volume in bottles was only performed for 60 % of the loaded bottles. Among these, 30 % contained the recommended volume of blood (between 7 and 13 ml). Finally, the contamination rate was found to be 27.2 % for samples at our institution.

Conclusions. The delays in sample registration, loading and unloading were found to be acceptable, even though they could be improved by ensuring a continuous service during the night duty period. Furthermore, the percentage of volumes measured is insufficient and must be improved and the majority of bottles do not contain the recommended blood volume.