- Volume 66, Issue 12, 2017

Purpose. Candida species have become resistant to commonly used anti-fungal drugs like fluconazole and echinocandins. In our screen, a series of quaternary ammonium compounds (QACs) emerged as an alternative treatment choice for drug-resistant Candida infections.

Methodology. Medium alkyl chain cationic lipo-oxazoles comprising six to thirteen twin carbon chains and a quaternary ammonium unit were synthesized and evaluated for their in vitro anti-Candida and biofilm inhibition activity. SEM was performed to visualize membrane distortion.

Results/Key findings. Heptyl and octyl chain analogues (5c, 6b and 6c) showed promising anti-fungal activity. Compound 5c was active against both fluconazole-sensitive and resistant clinical isolates of Candida albicans as well as non-albicans Candida strains. 5c also inhibited the adhesion of C. albicans cells to a polystyrene surface and restricted biofilm formation. SEM further confirmed Candida cell membrane distortion by 5c.

Conclusion. A novel class of QACs, called cationic lipo-oxazoles, was tested and found to exhibit anti-fungal activity against planktonic cells as well as biofilms of Candida.

Purpose. Human parvovirus B19 (B19V) can cause anemia in immunocompromised patients. We aimed to investigate the presence of B19V in HIV+ adults with different CD4+ T cell counts, to recognise the frequency of B19V in these different conditions and its possible association with anemia.

Methodology. We studied B19V specific IgM, IgG and DNA in 98 HIV+ patients and in 52 healthy individuals. HIV load, CD4+ counts and haemoglobin level were also determined in the patients.

Results. No individual in the control group had detectable IgM, 41/52 (78.8 %) had IgG and 5/52 (9.6 %) had B19V DNA. Among HIV+ patients, we found 5/98 (5.1 %) IgM+, 66/98 (67.3 %) IgG+ and 15/98 (15.3 %) had B19V DNA (no significant differences between the two groups compared). Considering the CD4+ cell range in HIV patients, 37 had <200 CD4+ cells ml−1, 31 had 200–500, and 30 had >500. Anti-B19V IgG prevalence in patients with >500 CD4+ cells ml−1 was significantly higher than in the rest (P=0.004) and compared to the control (P=0.046). B19V DNA concentration was always <103 IU ml−1, including 5 healthy individuals and 15 HIV+ patients. There was no significant association between B19V IgM or DNA and anemia nor between B19V DNA and HIV load.

Conclusions. The results indicate that B19V is not a high-risk factor for anemia in adult HIV+ patients under HAART treatment. Further studies will contribute to elucidate the mechanisms and significance of B19V DNA prevalence/persistence in adults, independently of the CD4+ cell status.

Purpose. Carbapenem resistance in P. aeruginosa is increasing worldwide. In Brazil, SPM-1 is the main P. aeruginosa carbapenemase identified. Little is known about the virulence factor in SPM-1 clones.

Methodolgy. We describe a carbapenem-resistant P. aeruginosa bloodstream infection (CRPa-BSI) outbreak in a bone marrow transplant Unit (BMT). Twenty-nine CRPa-BSI cases were compared to 58 controls. Microbiological characteristics of isolates, such as sensitivity, carbapenemase gene PCR for P. aeruginosa, and PFGE are described, as well as the whole-genome sequence (WGS) of three strains.

Results/Key findings. The cultures from environmental and healthcare workers were negative. Some isolates harboured KPC and SPM. The WGS showed that the 03 strains belonged to ST277, presented the same mutations in outer membrane protein, efflux pump, and virulence genes such as those involved in adhesion, biofilm, quorum-sensing and the type III secretion system, but differ regarding the carbapenemase profile. A predominant clone-producing SPM harbouring Tn 4371 was identified and showed cross-transmission; no common source was found. Overall mortality rate among cases was 79 %. The first multivariate analysis model showed that neutropenia (P=0.018), GVHD prophylaxis (P=0.016) and prior use of carbapenems (P=0.0089) were associated with CRPa-BSI. However, when MASCC>21 points and platelets were added in the final multivariate analysis, only prior use of carbapenems remained as an independent risk factor for CRPa-BSI (P=0.043).

Conclusions. The predominant clone belonging to ST277 showed high mortality. Carbapenem use was the only risk factor associated with CRPa-BSI. This finding is a wake-up call for the need to improve management in BMT units.

Purpose. To investigate linezolid-resistance mechanisms in linezolid-nonsusceptible enterococci (LNSE) isolated from a tertiary hospital in Korea.

Methodology. Enterococcal isolates exhibiting linezolid MICs ≥4 mg l−1 that were isolated between December 2011 and May 2016 were investigated by PCR and sequencing for mutations in 23S rRNA or ribosomal proteins (L3, L4 and L22) and for the presence of cfr, cfr(B) and optrA genes.

Results/Key findings. Among 135 LNSE (87 Enterococcus faecium and 48 Enterococcus faecalis isolates), 39.1 % (34/87) of E. faecium and 18.8 % (9/48) of E. faecalis isolates were linezolid-resistant. The optrA carriage was the dominant mechanism in E. faecalis: 13 isolates, including 10 E. faecalis [70 % (7/10) linezolid-resistant and 30 % (3/10) linezolid-intermediate] and three E. faecium [33.3 % (1/3) linezolid-resistant and 66.7 % (2/3) linezolid-intermediate], contained the optrA gene. G2576T mutations in the 23S rRNA gene were detected only in E. faecium [14 isolates; 71.4 % (10/14) linezolid-resistant and 28.6 % (4/14) linezolid-intermediate]. One linezolid-intermediate E. faecium harboured a L22 protein alteration (Ser77Thr). No isolates contained cfr or cfr(B) genes and any L3 or L4 protein alterations. No genetic mechanism of resistance was identified for 67.6 % (23/34) of linezolid-resistant E. faecium.

Conclusion. A low rate of 23S rRNA mutations and the absence of known linezolid-resistance mechanisms in the majority of E. faecium isolates suggest regional differences in the mechanisms of linezolid resistance and the possibility of additional mechanisms.

Purpose. The genus Arcobacter includes bacteria that are considered emergent pathogens because they can produce infections in humans and animals. The most common symptoms are bloody and non-bloody persistent diarrhea but cases with abdominal cramps without diarrhea or asymptomatic cases have also been described as well as cases with bacteremia. The objective was to characterize Arcobacter clinical strains isolated from the faeces of patients from three Spanish hospitals.

Methodology. We have characterized 28 clinical strains (27 of A. butzleri and one of A. cryaerophilus) isolated from faeces, analysing their epidemiological relationship using the multilocus sequence typing (MLST) approach and screening them for their antibiotic susceptibility and for the presence of virulence genes.

Results/Key findings. Typing results showed that only one of the 28 identified sequence types (i.e. ST 2) was already present in the MLST database. The other 27 STs constituted new records because they included new alleles for five of the seven genes or new combinations of known alleles of the seven genes. All strains were positive for the ciaB virulence gene and sensitive to tetracycline. However, 7.4 % of the A. butzleri and A. cryaerophilus strains showed resistance to ciprofloxacin.

Conclusion. The fact that epidemiological unrelated strains show the same ST indicates that other techniques with higher resolution should be developed to effectively recognize the infection source. Resistance to ciprofloxacin, one of the antibiotics recommended for the treatment of Arcobacter intestinal infections, demonstrated in 10.7 % of the strains, indicates the importance of selecting the most appropriate effective treatment.

Purpose. In Danish hospitals, the number of infections caused by vancomycin-resistant Enterococcus faecium (VRE faecium) has dramatically increased in recent years. Hospital disinfectants are essential in eliminating pathogenic microorganisms, and reduced susceptibility may contribute to hospital-associated infections. We have addressed whether clinical VRE faecium display decreased biocide susceptibility when compared to vancomycin-sensitive Enterococcus faecium (VSE faecium) isolates.

Methodology. In total 12 VSE faecium and 37 VRE faecium isolates obtained from Danish hospitals over an extended time period were tested for susceptibility towards three commonly applied biocides, namely benzalkonium chloride, chlorhexidine and hydrogen peroxide.

Results. For benzalkonium chloride, 89 % of VRE faecium strains had a minimal inhibitory concentration (MIC) of 8 mg l−1, whereas for VSE faecium, only 25 % of the strains had an MIC of 8 mg l−1. For chlorhexidine, the MIC of 95 % of VRE faecium strains was 4 mg l−1 or higher, while only 33 % of VSE faecium strains displayed MIC values at the same level. In contrast, both VRE and VSE faecium displayed equal susceptibility to hydrogen peroxide, but a higher minimal bactericidal concentration (MBC) was found for the former. The efflux activity was also assessed, and this was generally higher for the VRE faecium strains compared to VSE faecium.

Conclusion. VRE faecium from Danish hospitals demonstrated decreased susceptibility towards benzalkonium chloride and chlorhexidine compared to VSE faecium, where the use of chlorhexidine is particularly heavy in the hospital environment. These findings suggest that biocide tolerance may characterize VRE faecium isolated in Danish hospitals.

Purpose. Fast identification of bacteria directly from positive blood cultures (BCs) by matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF MS) can be achieved either using the MALDI Sepsityper kit (protein extraction method) or after a short-term pre-cultivation step on solid medium. We developed a new method that involves short-term enrichment of positive BCs in brain–heart infusion broth (BHI) prior to MALDI-TOF MS analysis.

Methodology. Eighty-four BCs flagged as positive were included in this study; these were processed in parallel either directly using the MALDI Sepsityper kit or following a short-term culture either in BHI or on Columbia blood agar with 5 % sheep blood (CBA).

Results. Bacterial species were successfully identified in 91.6, 89.2 and 65.4 % of cases after pre-cultivation for 4 h in BHI, on CBA, or by using the MALDI Sepsityper kit, respectively. Overall, the mean incubation time to correct identification was shorter when pre-cultures were performed in BHI; the mean time for Gram-negative rods was 78.2 min in BHI and 108.2 min on CBA (P=0.045), and the mean time for Gram-positive cocci was 128.5 min in BHI and 169.6 min on CBA (P=0.013).

Conclusion. Short-term enrichment of BCs in BHI accelerates identification of a number of bacterial species by MALDI-TOF MS. Further prospective studies are needed to validate our method and gauge its potential clinical impact on the management of bloodstream bacterial infections.

Purpose. Fungaemia is associated with substantial morbidity and mortality in neonates admitted to neonatal intensive care units (NICUs). We report an outbreak of fungaemia in a NICU due to rare yeast, Pichia kudriavzevii (a teleomorph of Candida krusei). To the best of our knowledge, this is the first report of neonatal sepsis due to P. kudriavzevii.

Methodology. Between August and September 2014, blood cultures from nine neonates diagnosed with late-onset sepsis in the NICU yielded yeast-like organisms. The molecular identification and typing of these isolates was performed by sequencing the D1/D2 region of 26S rDNA and fluorescent amplified fragment length polymorphism (FAFLP) respectively. Antifungal susceptibility was tested by broth microdilution as per the Clinical Laboratory Standards Institute (CLSI) guidelines. Sampling from environmental sources and the hands of healthcare workers (HCWs) in the NICU was performed.

Results. Of the nine neonates, eight were preterm and six had very low birth weight (VLBW). Thrombocytopenia was present in two neonates. Sequencing identified all the isolates as P. kudriavzevii and FAFLP showed their clonal origin. Antifungal susceptibility testing revealed the susceptibility of all isolates to the antifungals tested. Treatment with voriconazole was advised. However, only seven neonates were treated successfully and discharged after improvement, whereas two were lost for follow-up. Cultures from the environment and the hands of HCWs were negative. The outbreak was controlled by the strict implementation of infection control practices.

Conclusion. This study emphasizes the importance of accurate identification of the aetiological agent of sepsis and vigilant monitoring for the possibility of an outbreak in NICUs.

Purpose. Data regarding the incidence and molecular epidemiology of Clostridium difficile infections (CDIs) in Greece are limited.

Methodology. A retrospective study of all laboratory-confirmed CDI cases in a university hospital during a 9-month period. Stool samples from inpatients with diarrhoea were tested with a combined glutamate dehydrogenase (GDH) and toxin enzyme immunoassay (EIA) test, as part of a two-step algorithm for CDI testing. All GDH-positive samples were cultured and isolates were further tested for the presence of toxin genes and characterized by PCR ribotyping.

Results. The incidence of CDI in our hospital was 25 per 10 000 hospital admissions. Of 33 CDI cases, 72.7 % were hospital-acquired. Fourteen different PCR ribotypes were identified, of which 017 (21.2 %), 078/126 (15.1 %) and RT202 and RT106 (9 %) were the most prevalent. Most patients had a risk profile of recent antibiotic use, older age and comorbidities. Despite mild CDI clinical characteristics, six cases showed complications and led to 18.2 % mortality.

Conclusion. The CDI incidence was comparable to that in other European countries. The hypervirulent PCR ribotype 027 was not found, whereas ribotypes 017 and 126 predominated. Most CDI cases were in patients who used antibiotics, emphasizing that antimicrobial stewardship should be considered as a cornerstone for the prevention of CDI.

Purpose. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of nosocomial infections. A thorough understanding of the epidemiology and distribution of MRSA allows the development of better preventive measures and helps to control or reduce the rate of infection among the general population.

Methodology. A retrospective survey was performed on 511 cases of MRSA infections from inpatient, outpatient and nursing home populations over a 12-month period. To study the relationships between two continuous quantitative variables (patient age vs resistance percentage), a simple linear regression was calculated for each antibiotic to predict the antibiotic resistance percentage with respect to patient age.

Results/Key findings. The pattern of antibiotic resistance with respect to the age of patients depended on the antibiotic mode of action. Antibiotics that target DNA synthesis (i.e. fluoroquinolones) display a direct correlation with the age of patients, with higher rates of resistance among the older population, while antibiotics that target ribosomal functions (i.e. aminoglycosides) or cell wall synthesis (i.e. cephalosporin) do not display an age-dependent pattern and have a consistent degree of resistance across all age classes.

Conclusion. Antibiotics that target DNA synthesis result in a progressively higher number of resistant isolates among the older population. The results emphasize the importance of patient age on antibiotic selection as a preventive measure to reduce the rate of resistant infections in each susceptible population. This pattern suggests that physicians should take into consideration patient age as another factor in determining the best antibiotic regiment with the aim of curtailing the emergence of newer resistant phenotypes in the future.

Purpose. Enzootic bovine leucosis (EBL) is a silent disease caused by a retrovirus [bovine leukaemia virus (BLV)]. BLV is classified into almost 10 genotypes that are distributed in several countries. The present research aimed to describe two BLV gp51 env sequences of strains detected in the state of São Paulo, Brazil and perform a phylogenetic analysis to compare them to other BLV gp51 env sequences of strains around the world.

Methodology. Two bovines from different herds were admitted to the Bovine and Small Ruminant Hospital, School of Veterinary Medicine and Animal Science, University of São Paulo, Brazil. In both, lymphosarcoma was detected and the presence of BLV was confirmed by nested PCR. The neighbour-joining algorithm distance method was used to genotype the BLV sequences by phylogenetic reconstruction, and the maximum likelihood method was used for the phylogenetic reconstruction. The phylogeny estimates were calculated by performing 1000 bootstrap replicates.

Results. Analysis of the partial envelope glycoprotein (env) gene sequences from two isolates (25 and 31) revealed two different genotypes of BLV. Isolate 25 clustered with ten genotype 6 isolates from Brazil, Argentina, Thailand and Paraguay. On the other hand, isolate 31 clustered with two genotype 5 isolates (one was also from São Paulo and one was from Costa Rica). The detected genotypes corroborate the results of previous studies conducted in the state of São Paulo, Brazil. The prediction of amino acids showed substitutions, particularly between positions 136 and 150 in 11 out of 13 sequences analysed, including sequences from GenBank.

Conclusion. BLV is still important in Brazil and this research should be continued.