- Volume 68, Issue 9, 2019

Introduction. Non-photochromogenic rapidly growing mycobacteria (NPRGM) that branch distinctly from Mycobacteroides (Myco) and Mycolicibacterium (Mycolici) are increasingly observed clinically and present a complicated treatment challenge; thus, appropriate in vitro susceptibility testing is required.

Methodology. We evaluated the MICs of nine antimicrobials used in the treatment of infections of 25 NPRGM type strains. The relation of inducible macrolide resistance with functional erythromycin ribosomal methylase (erm) genes was also investigated.

Results. The initial clarithromycin MIC reading at 3 days showed resistance in four of the Mycolici strains. In contrast, the presence of erm genes among Mycolici species differed from previous findings. Both Myco and Mycolici species were highly susceptible to amikacin and linezolid. Myco species were resistant to fluoroquinolones, while Mycolici species were susceptible. Meropenem showed low activity against Myco species, but high activity against Mycolici species.

Conclusion. NPRGM clade-specific susceptibility patterns suggest an urgent need to establish distinct breakpoints for Myco and Mycolici species.

The molecular mechanism of Helicobacter pylori resistance to tetracycline involves mutations in the primary binding site of the ribosome. A resistance or reduced susceptibility to tetracycline could be the result of single, double or triple mutations in the 16S rRNA gene of H. pylori . We investigated if the genotype was correlated to tetracycline resistance as determined phenotypically in vitro for 96  H . pylori isolates in the gastroesophageal mucosa of Venezuelan individual hosts. E-test for antimicrobial susceptibility test and real-time PCR for the detection of 16S rRNA gene mutations were performed in 96  H . pylori isolates (48 obtained from antrum, and 48 from oesophagus) from eight dyspeptic patients. In the gastric mucosa, 38 isolates were identified sensitive and 10 resistant to tetracycline by E-test, whereas 44 sensitive and 4 resistant isolates were found in the oesophagus. Real-time PCR detection of the 16S rRNA gene exhibited mutants with a single base-pair substitution (AGA926 GGA) in six antrum isolates and seven oesophagus isolates, whereas only three harboured a low level of tetracycline resistance in vitro. Our results indicate that real-time PCR detection of 16S rRNA is a reliable method to classify among tetracycline-resistant genotypes and useful in patients who have experienced a first-line treatment failure with triple therapy.

Introduction. Carbapenemase-producing Enterobacteriaceae have become a major public health concern over the last decade and treatment options are limited.

Aims. We evaluated the synergistic activity of the combination of aztreonam (ATM) and clavulanate for 41 β-lactam-resistant clinical isolates harbouring class B or/and class D carbapenemases combined or not with extended-spectrum β-lactamases (ESBLs).

Methodology. The MICs of ATM, with and without amoxicillin–clavulanate (AMC), were determined. Time-kill assays were performed for three representative strains.

Results. The ATM–AMC combination had a synergistic effect on 34/41 (83 %) isolates. The MIC of ATM, in the presence of clavulanate, was ≤1 mg l−1 for 15/41 (37 %) isolates and ≤4 mg l−1 for 29/41 (71 %) isolates. Synergistic activity was observed for 34/37 (92 %) isolates producing ESBLs and carbapenemases, compared to 0/4 (0 %) for ESBL-negative strains. Complete or partial bactericidal activity was obtained when the MIC of the combination was 0.5 mg l−1 and 1.5 mg l−1 or 8 mg l−1, respectively.

Conclusion. The combination of ATM and AMC could be an attractive unconventional treatment for infections due to carbapenemase- and ESBL-producing Enterobacteriaceae.

Introduction. Umbilical catheterization offers unique vascular access that is only possible in the neonatal setting due to unobstructed umbilical vessels from foetal circulation. With the cut of the umbilical cord, two arteries and a vein are dissected, allowing quick and painless catheterization of the neonate. Unfortunately, keeping the umbilical access sterile is challenging due to its mobility and necrosis of the umbilical stump, which makes it a perfect model for vessel catheter colonization analysis.

Aim. The aim of this study was to evaluate bacterial colonization of the umbilical catheter, with a focus on the difference between various sections of the catheter, the duration of catheterization, patient status and gestational age.

Methodology. We performed bacterial cultures for 44 umbilical catheters, analysing the superficial and deep parts of the catheter separately, and revealed colonization in one-third of cases.

Results. One hundred per cent of the colonization occurred in preterm infants, with a shift towards extreme prematurity. The catheters were mainly colonized by coagulase-negative staphylococci. The majority of catheters presented with superficial colonization dominance, and there were no cases of deep colonization. The bacterial strains and their resistance were consistent between the catheter’s proximal and distal parts, as well as positive blood cultures. The patients with the most intense bacterial catheter colonization presented with sepsis around removal time or a couple of days later, especially if they were extremely premature and exhibited very low birth weight. Catheterization time did not play a major role.

Conclusion. Umbilical catheters are vectors for skin microflora transmission to the bloodstream via biofilm formation, regardless of antibiotic use and the duration of catheterization, especially in preterm neonates.

Introduction. Current intradermal tuberculin skin tests for latent tuberculosis infection (LTBI) based on purified protein derivative (PPD) have poor specificity.

Aims. Developing a better skin test antigen as well as a simple skin patch test may improve and facilitate diagnostic performance.

Methodology. Defined recombinant antigens that were unique to Mycobacterium tuberculosis (MTB), including two potential latency-associated antigens (ESAT-6 and Rv2653c) and five DosR-encoded latency proteins (Rv1996, Rv2031c, Rv2032, DevR and Rv3716c), were used as diagnostic skin test reagents in comparison with a standard PPD. The performance of the skin tests based on the detection of delayed-type hypersensitivity (DTH) reaction in guinea pigs sensitized to MTB and M. bovis bacille Calmette–Guérin (BCG) vaccine was evaluated.

Results. The latency antigens Rv1996, Rv2031c, Rv2032 and Rv2653c and the ESAT-6 protein elicited less reactive DTH skin responses in MTB-sensitized guinea pigs than those resulting from PPD, but elicited no response in BCG-vaccinated guinea pigs. The remaining two latency antigens (DevR and Rv3716c) elicited DTH responses in both groups of animals, as did PPD. The reactivity of PPD in BCG-vaccinated guinea pigs was greater than that of any of the selected skin test reagents. Using stronger concentrations of selected skin test reagents in the patch test led to increased DTH responses that were comparable to those elicited by PPD in guinea pigs sensitized with MTB.

Conclusion. Transdermal application of defined purified antigens might be a promising method for LTBI screening.

The recent increase in pertussis cases observed in some countries may have several causes, including the evolution of Bordetella pertussis populations towards escape of vaccine-induced immunity. Most genomic studies of B. pertussis isolates performed so far are from countries that use acellular vaccines. The objective was to analyse genomic sequences of isolates collected during the 2014 whooping cough epidemic in Tunisia, a country where whole-cell vaccines are used. Ten Tunisian isolates and four vaccine strains were sequenced and compared to 169 isolates from countries where acellular vaccines are used. Phylogenetic analysis showed that Tunisian isolates are diverse, demonstrating a multi-strain 2014 epidemic peak, and are intermixed with those circulating in other world regions, showing inter-country transmission. Consistently, Tunisian isolates have antigen variant composition observed in other world regions. No pertactin-deficient strain was observed. The Tunisian B. pertussis population appears to be largely connected with populations from other countries.

Purpose. To investigate the use of a corneal impression membrane (CIM) for the detection of herpes simplex virus type 1 (HSV-1) in suspected herpes simplex keratitis (HSK).

Methodology. In the laboratory study, swabs and CIMs made from polytetrafluoroethylene were spiked with different concentrations of HSV-1. DNA was extracted and real-time PCR undertaken using two sets of primers. In the clinical study, consecutive patients presenting with suspected HSK were included. For each patient, samples were collected from corneal lesions with a swab and a CIM in random order. Clinical details were collected using a standardized clinical form and patients were categorized into probable, presumed and possible HSK.

Results. There was no difference in the performance of both primer sets for all HSV-1 dilutions (P=0.83) using a CIM or between a CIM and a swab (P=0.18). In total, 110 patients were included. Overall, 73 patients (66.4 %) had probable, 20 patients (18.2 %) presumed and 17 patients (15.5 %) possible HSV-1 keratitis. The HSV-1 detection rate was significantly higher using a CIM (40/110, 36.4 %) than a swab (28/110, 25.5 %) (P=0.004). In the probable HSV keratitis group, the detection rate using a CIM was 43.8 % compared to 27.4 % for a swab (P=0.004). The cycle threshold values obtained for the conjunctival swabs were higher than those obtained for the CIMs (P<0.001).

Conclusions. In suspected HSK, a CIM is a useful alternative to a swab and more likely to detect the presence of HSV-1.

Purpose. This study aimed to characterize 27 Escherichia coli isolates obtained from peritoneal dialysis (PD)-related peritonitis that occurred at the University Hospital of Botucatu Medical School, Brazil, between 1997 and 2015.

Methodology. These isolates were characterized regarding the occurrence of 22 virulence factor-encoding genes, antimicrobial resistance and biofilm production. We then evaluated whether these factors influenced the clinical outcome.

Results. Over an 18-year period, 726 episodes of PD-related peritonitis were diagnosed, with 27 of them (3.7 %) being due to E. coli . The majority of the isolates were classified in phylogroups B1 (33.3 %), B2 (30.0 %) or F (18.0 %). fimH (100.0 %), ompT (66.7 %) and irp2 (51.9 %) were the most prevalent genes, while papA, papC, iha, sat, irp2, iucD, ireA, ibe10, ompT and kpsMTII were significantly more prevalent among isolates belonging to phylogroups B2 and F (P<0.05). Non-susceptibility to quinolones was detected in six isolates, which harboured chromosomal and/or plasmid-mediated quinolone resistance determinants, while two CTX-M extended-spectrum β-lactamase-producing E. coli were identified. Virulence factor-encoding genes (alone or in combination) and antimicrobial resistance were not associated with non-resolution outcomes. However, there was a trend for the ability to produce biofilm to be associated with treatment failure, although this association was not statistically significant.

Conclusion. The E. coli isolates were heterogeneous in terms of the features investigated, and were susceptible to most of the antimicrobial drugs tested, despite the unsuccessful treatment observed in more than 50.0 % of the patients. Studies including more cases could help to clarify if biofilm production can influence the outcome in patients with PD-related peritonitis.

Introduction. Bacterial vaginosis (BV) is dysbiosis associated with an increased risk of several sexually transmitted infections. It is primarily diagnosed via Gram staining, although molecular analyses have presented higher diagnostic accuracy.

Aim. This study aimed to evaluate the molecular epidemiology of BV in asymptomatic women to determine its association with several commensal and pathogenic micro-organisms of the genitalia.

Methodology. The prevalence of BV was investigated through semiquantitative assessment of 201 women recruited during their routine gynaecological inspection at an outpatient clinic in Tabasco, Mexico.

Results. Women with BV showed an increased prevalence of Chlamydia trachomatis (P=0.021) and Mycoplasma hominis (P=0.001). Of the BV-associated micro-organisms, Gardnerella vaginalis was significantly associated with C. trachomatis (P=0.005) and/or Ureaplasma parvum (P=0.003), whereas Atopobium vaginae and Megasphaera type 1 correlated significantly with Mycoplasma hominis (P=0.001). No significant association was observed between human papillomavirus (HPV) infection and BV, although there was increased prevalence of HPV59, HPV73, HPV52 and HPV58 in women displaying cervical cytological abnormalities.

Conclusion. Identification of BV-associated micro-organisms via molecular analysis may help to distinguish recurrent cases from new infections and identify micro-organisms potentially associated with pharmacological resistance.

Introduction. In 2016–2017, there was an increase in the number of paediatric invasive pneumococcal disease (IPD) cases caused by Streptococcus pneumoniae serotype 12F in Chiba Prefecture, Japan. Serotype 12F is one of the major causative serotypes of IPD following the introduction of pneumococcal conjugate vaccine 13 (PCV13), and outbreaks of IPD caused by serotype 12F have recently been reported in several countries.

Aim. Our goal here was to clarify the relationship among local outbreak strains and the outbreak strains in other countries, and for this we analysed clinical isolates of S. pneumoniae serotype 12F using several genetic identification methods.

Methodology. All reported IPD cases caused by serotype 12F were reviewed and bacterial strains were collected and analysed. We also analysed S. pneumoniae serotype 12F strains isolated from other time periods, geographical areas, cases of adult IPD and respiratory specimens as control strains. Multi-locus sequence typing, PFGE and multi-locus variable number tandem repeat analysis (MLVA) were conducted on all isolates.

Results. All 26  S . pneumoniae serotype 12F isolates, including control strains, belonged to a single sequence type (ST4846) that was the specific ST in Japan. All tested strains demonstrated five MLVA patterns and two PFGE patterns.

Conclusion. We determined that the 2016–2017 outbreak of IPD in Chiba Prefecture was caused by clonally related isolates of serotype 12F. The continuous monitoring of IPD caused by serotype 12F is important for evaluating the impact of re-emerging pneumococcal serotypes following the PCV13 introduction era, and MLVA could be a useful tool for identification of outbreak strains.