- Volume 68, Issue 1, 2019

There are challenges regarding increased global rates of microbial resistance and the emergence of new mechanisms that result in microorganisms becoming resistant to antimicrobial drugs. Fosfomycin is a broad-spectrum bactericidal antibiotic effective against Gram-negative and certain Gram-positive bacteria, such as Staphylococci, that interfere with cell wall synthesis. During the last 40 years, fosfomycin has been evaluated in a wide range of applications and fields. Although numerous studies have been done in this area, there remains limited information regarding the prevalence of resistance. Therefore, in this review, we focus on the available data concerning the mechanisms and increasing resistance regarding fosfomycin.

Purpose. Bloodstream infections are major causes of morbidity and mortality among hospitalized patients worldwide. Early identification of micro-organisms from blood culture can facilitate earlier optimization of treatment. The objective of this study was to assess an in-house method based on a new matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform (Clin-TOF MS) for direct organism identification.

Methodology. We studied the performance of the in-house method for direct identification and the conventional sub-culture method in parallel. Identification from subcultures was analysed with Bruker MS as the reference method.

Results. A total of 666 blood cultures with a single micro-organism that flagged positive after no more than a 3-day incubation period were collected. The identification accuracy of the in-house Clin-TOF MS method for direct identification and the sub-culture method was 88.6 and 100 %, respectively. The in-house method exhibited better performance for Gram-negative bacteria than for Gram-positive bacteria (93.3 vs 81.6 %). The accuracy rate for anaerobes was 100 % (3/3). The lowest accurate identification rate was for yeast; this was only 20 %. Lytic Anaerobic/F (LAF) and Plus Aerobic/F (PAF) provided the highest accurate identification rates, and it was noteworthy that the accuracy rate for FAN Aerobic (FA) was 82 %, which is higher than previously reported and showed that the method was effective.

Conclusion. Our study provides an effective sample preparation method for the direct identification of pathogens from positive blood culture vials via Clin-TOF MS at a very low cost of about $0.5 per sample and with a short turnaround time of about 20 min. This will help clinicians make precise diagnoses and provide targeted prescriptions, reducing the risk of the potential development of resistance.

A sharp increase in staphylococcal scalded skin syndrome (SSSS) cases has been recorded in our settings since 2015, with 31 cases having been documented during the period 2014–2017. The molecular investigation of strains from the above period showed the emergence of a methicillin-susceptible, mupirocin- and fusidic acid-resistant Staphyloccocus aureus clone that belongs to the ST121 complex and carries both epidermolysin (eta/etb) genes. We concluded that the SSSS caused by the newly emerged, highly virulent community-associated-methicillin sensitive S. aureus strains that have been encountered lately is more severe than impetigo. Physicians should be aware of the probability of SSSS epidemics from strains that are resistant to mupirocin and fusidic acid, which have been used irrationally and excessively.

Objectives. Acinetobacter baumannii is a critical nosocomial pathogen. A. baumannii infections have become a grave challenge due to their ability to develop resistance to different antimicrobial agents. The current study aimed to evaluate the potential synergism and bactericidal activity of a combination of colistin and cotrimoxazole against carbapenem-resistant A. baumannii (CRAB) in a Galleria mellonella model.

Methods. Four clinical A. baumannii isolates were biochemically and molecularly identified. Their antimicrobial susceptibility levels were established and the molecular characterization of the carbapenemase-encoding genes was performed. The synergism and bactericidal effect of the colistin/cotrimoxazole combination was assessed using the checkerboard assay and time–kill experiments. An in vivo evaluation of the activity of the combination was performed using the Galleria mellonella model.

Results. A fractional inhibitory concentration index (FICI) of ≤0.5 was found for all strains, indicating that the colistin/cotrimoxazole combination exhibited powerful synergistic activity. The combination displayed both synergistic and bactericidal activity at sub-breakpoint concentrations for all strains. Cotrimoxazole monotherapy showed the least protective activity in the G. mellonella model. The survival rate ranged from 66.7–79.2 % at 24 h and was 29.2–60.4 % at 96 h for the tested isolates. Colistin monotherapy performed better than cotrimoxazole monotherapy; the G. mellonella survival rate ranged from 77.1–97.9 %, at 24 h and from 64.5–72. % at 96 h. The colistin/cotrimoxazole combination improved G. mellonella’s survival rate at 96 h remarkably in comparison to colistin or cotrimoxazole monotherapy.

Conclusions. Finally, the combination of colistin and cotrimoxazole appears to be a promising therapeutic option for the management of CRAB-associated infections. It is essential to assess the clinical application and the dose–response relationships of combinations such as colistin plus cotrimoxazole.

Purpose. Despite being in clinical use for decades, colistin susceptibility testing remains challenging because of its inherent cationic properties. We aimed to compare the performance characteristics of different methods for testing susceptibility to colistin in a series of clinical isolates of Gram-negative bacilli.

Methodology. One hundred and nine clinical isolates of Klebsiella pneumoniae (n=34), Escherichia coli (n=20), Acinetobacter baumannii (n=17) and Pseudomonas aeruginosa (n=38) were studied for colistin susceptibility using broth microdilution (BMID), broth macrodilution (BMAD), agar dilution (AD) as well as disc-diffusion (DD) utilizing two different commercial disc sources.

Results. By using BMID as reference method, 88 and 21 isolates were found to be colistin susceptible and resistant, respectively. Overall, acceptable essential agreement (EA) and categorical agreement (CA) were observed between BMAD and reference method (100 %). Whereas the AD method revealed the lowest rate of EA (61.7, 11.7, 5.0 and 5.2 % for K. pneumoniae, A. baumannii, E. coli and P. aeruginosa, respectively), it showed acceptable or near acceptable CA for K. pneumoniae (100 %), E. coli (100 %) and A. baumannii (88.2 %) isolates but not for P. aeruginosa (13.1 %). DD failed to detect resistance in colistin-resistant (colR) P. aeruginosa (n=5, very major errors of 100 %) but successfully identified all high-level colistin-resistant A. baumannii and K. pneumoniae isolates.

Conclusion. We found BMAD to be very reliable for colistin MIC determination. Methods AD and DD should not be used for colistin susceptibility testing in P. aeruginosa isolates as these are associated with false-resistant and -susceptible results, respectively.

Purpose. Multidrug-resistant Klebsiella pneumoniae strains are regularly involved in hospital outbreaks. This study describes an ESBL-producing K. pneumoniae clone (ST607-K25) responsible for a nosocomial outbreak in a neonatal intensive care unit.

Methodology. Fourteen strains isolated from 13 patients were included. Antimicrobial susceptibility testing was performed by the agar diffusion method. A clonal link was first investigated by fingerprinting (ERIC-PCR and REP-PCR) then confirmed by MLST. Characterization was performed by molecular detection and identification of several drug resistance and virulence determinants.

Results. All strains expressed the same antibiotype, combining ESBL production, fluoroquinolones and aminoglycoside resistance, except for one which remained susceptible to fluoroquinolones. Fingerprinting methods confirmed the clonal link and MLST identified a ST607 clone. Molecular investigations revealed: (I) genes encoding for two narrow-spectrum beta-lactamases (SHV-1 and TEM-1) and an ESBL (CTX-M-15); (II) absence of any chromosomal mutation in quinolone resistance-determining- regions (QRDR) of gyrA/gyrB and parC/parE genes; (III) genes encoding for three plasmid-mediated quinolone-resistance (PMQR) determinants: oqxAB (14/14), aac(6′)-Ib-cr (14/14) and qnrB (13/14); (IV) production of a K25 capsule; and (V) carriage of three genes encoding for virulence factors: mrkD (type 3 fimbriae) (14/14), ybts (yersiniabactin) (12/14) and entB (enterobactin) (14/14).

Conclusion. We described a multidrug-resistant Kp ST607 clone responsible for a nosocomial outbreak in vulnerable and premature newborns. Molecular investigations allowed us to identify several resistance factors responsible for ESBL production (CTX-M-15) and quinolone resistance (three PMQR determinants). The detection of a gene (ybtS) belonging to the high-pathogenicity island yersiniabactin could partly explain its high colonization and diffusion potential.

Purpose. Tissue samples from patients with suspicion of deep or subcutaneous fungal infections were analysed at the Portuguese Reference Mycology Laboratory according to a proposed diagnostic approach, which aims to constitute a rapid and accurate diagnosis for these fungal infections.

Methodology. Forty-six tissue biopsy samples were analysed over a period of 26 months, using a diagnostic approach that includes culture, panfungal PCR and Aspergillus-directed PCR.

Results/Key findings. Overall, 23 samples were reported as negative while the remaining 23 were reported as positive for fungi (PCR, culture and/or histology). PCR showed an estimated detection limit of 12 pg DNA µl–1. From the 46 samples, 30 were negative for fungal DNA while 16 gave positive results. From these, 12 cases were detected by panfungal PCR and six cases by PCR directed toward Aspergillus. In 61 % of the cases, there was concordance between molecular and cultural methods. Aetiological agents identified were Candida albicans, C. glabrata, C. tropicalis, Trichosporon montevideense, Alternaria spp., Exophiala sp., Trichoderma sp., Histoplasma spp., Aspergillus fumigatus, Trichophyton rubrum and Paracoccidioides brasiliensis.

Conclusion. Our results showed that the proposed polyphasic approach appears to be a useful strategy in the detection of fungi from tissue samples, allowing a better prognosis. In further studies, the inclusion of a higher number of samples and the implementation of more genus-specific PCRs will certainly contribute to an increase in the specificity and sensitivity of this method.

Sporadic diphtheria cases in Malaysia have remained low in number since the 1990s. However, in 2016 a total of 31 cases were reported nationwide and to investigate this we performed molecular characterization of 30 Corynebacterium diphtheriae isolates collected from 1981 to 2016 using multilocus sequence typing (MLST). C. diphtheriae isolates were identified and biotyped using the API Coryne kit, while the toxigenicity was determined by PCR and the Elek test. All of the 2016 isolates belonged to biotype mitis, caused respiratory diphtheria and were toxigenic strains. MLST analysis identified 17 sequence types (STs), including 11 new ones. ST453 was the most common clone (7/30, 23.3 %), followed by ST141 (5/30, 16.7 %), ST451 (3/30, 10.0 %) and ST248 (2/30, 6.7 %). The clones identified in 2016 had not been detected in previous isolations and they were phylogenetically distinct. Our results suggest that the diphtheria cases in 2016 were caused by the emergence and spread of new clones in Malaysia.