Phenotypic and molecular characterization of macrolide resistance mechanisms among Streptococcus pneumoniae isolated in Tunisia.

Introduction. Streptococcus pneumoniae is responsible for many community infections, with the main ones being pneumonia and meningitis. Pneumococcus has developed increased resistance to multiple classes of antibiotics. The evolution of antibiotic resistance in pneumococcus was influenced by changes in serotype distribution under vaccine selection pressure.Aim. The aim of this study was to determine the genes involved in macrolide resistance, the antimicrobial susceptibility, the serotype distribution and the spread of international antibiotic-resistant clones among clinical isolates of S. pneumoniae.Methodology. We investigated 86 erythromycin-resistant S. pneumoniae strains isolated from respiratory (n=74) or non-respiratory (n=12) samples in Tunisia. Antimicrobial susceptibility was tested using the disk diffusion method. Macrolide-resistant strains were analysed by polymerase chain reaction (PCR) for ermA, ermB, mefA and msrD. We also investigated the macrolide resistance mechanisms in eight isolates (9.3%) by sequencing the L4 and L22 riboprotein-coding genes, plus relevant segments of the three 23S rRNA genes. Capsular serotypes were detected by multiplex PCR. Sequence types (STs) were explored using multilocus sequence typing (MLST).Results. Among the 86 studied strains, 70 (81.4 %) were resistant to penicillin G. The prevalent serotypes were 19F, 14, 19A and 23F. We observed that the cMLSB phenotype (66/86, 76.7%) was the most common in these pneumococci. In addition, ermB was the most frequent resistance gene. No mutation in ribosomal protein L22 or L4 or 23S rRNA was detected. Overall, 44 STs were identified in this study, including 16 that were described for the first time. Resistance to lincomycin, tetracycline and trimethoprim/sulfamethoxazole was observed in 55 (64 %), 34 (39.5 %) and 31 (36 %) isolates, respectively. Furthermore, an increase in fluoroquinolone use in particular may lead to the emergence of levofloxacin-resistant strains. Multidrug resistance was observed in 83 isolates (96.5%). Three global antibiotic-resistant clones were identified: Denmark14 ST230, Portugal19F ST177 and Spain9V ST156.Conclusion. This study shows that macrolide resistance among S. pneumoniae isolated in Tunisia is mainly related to target site modification. Our observations demonstrate a high degree of genetic diversity and capsular types among strains resistant to macrolides.

Genetic diversity of Streptococcus pneumoniae in Tunisia.

OBJECTIVES: This study aimed to explore the genetic diversity of Streptococcus pneumoniae isolates in a Tunisian pneumology hospital. METHODS: A total of 141 S. pneumoniae strains isolated between 2009-2016 in the microbiology laboratory at A. Mami Hospital of Pneumology were investigated. Antimicrobial susceptibility testing was performed the disk diffusion method. MICs of penicillin G, amoxicillin and cefotaxime were determined by Etest. Serotyping was inferred from the results of multiplex PCR targeting 40 serotypes. Sequence types (STs) were determined by multilocus sequence typing (MLST). RESULTS: Among the 141 S. pneumoniae isolates, 98 (69.5%) were resistant to erythromycin. Evaluation of ß-lactam susceptibility showed that 90 strains (63.8%) were non-susceptible to penicillin, whereas 48 (34.0%) had decreased susceptibility to amoxicillin and 21 (14.9%) to cefotaxime. Twenty-five serotypes were detected, and 10 isolates were classified as non-typeable. Vaccine coverage was 56.7%, 60.3% and 75.2% for pneumococcal conjugate vaccine 7 (PCV7), PCV10 and PCV13, respectively. Overall, 73 STs were identified, including 23 described for the first time. The most frequent STs were ST179 (n = 17), ST3772 (n = 14), ST2918 (n = 10) and ST4003 (n = 5), related to serotypes 19F, 19A, 14 and 23F, respectively. Moreover, 110 strains were classified within 45 STs. Three international antimicrobial-resistant clones were found, including Denmark14-ST230 (n = 22), Spain9V-ST156 (n = 22) and Portugal19F-ST177 (n = 20). CONCLUSION: This study emphasises the clonal and international dissemination of antimicrobial-resistant S. pneumoniae clones. Significant differences in genetic variation were documented by MLST within the various serotypes identified.

Is sequencing better than phenotypic tests for the detection of pyrazinamide resistance?

SETTING: Phenotypic tests used to detect pyrazinamide (PZA) resistance are slow and have a high rate of false resistance. OBJECTIVE: To evaluate the accuracy of pncA sequencing for the detection of PZA resistance in Mycobacterium tuberculosis strains isolated in Tunisia. DESIGN: A total of 82 isolates, 41 resistant and 41 susceptible to PZA on BACTEC™ MGIT™ 960, were sequenced for pncA. Whole genome sequencing was performed for strains that were phenotypically resistant and had wild-type pncA in addition to MGIT retesting with a modified protocol. RESULTS: Twenty-three strains resistant to PZA with negative pyrazinamidase (PZase) activity harboured a mutation in the promoter or coding region of pncA. However, 18 strains resistant to PZA did not present any mutation. Repeat MGIT 960 showed that 16 of 18 M. tuberculosis isolates were falsely resistant to PZA. Compared with MGIT, PZase activity assay and pncA sequencing both presented a sensitivity of 92.0% (95%CI 73.9-99.0) and a specificity of respectively 96.5% (positive predictive value [PPV] 92.0%, negative predictive value [NPV] 96.5%) and 100.0% (PPV 100.0%, NPV 96.6%). CONCLUSION: The standard MGIT assay showed a high rate of false resistance to PZA, and the PZase activity assay is slow. pncA sequencing could therefore represent a rapid, accurate, alternative test to detect PZA resistance.

[New methods of diagnosis in tuberculosis]. / Nouvelles méthodes de diagnostic de la tuberculose.

Bacteriological diagnosis of tuberculosis has benefited in recent years from many technological advances to improve rapidity and sensitivity of the techniques. Thus, new LED fluorescence microscopes are in the process of replacing the optical microscopes and the Ziehl-Neelsen technique, making the examination more precise, faster and easier. The manual and automatic liquid culture has improved Lowenstein-Jensen culture and helped shorten antibiotic sensitivity test, allowing appropriate management of patients. The development and standardization of molecular biology methods led to the rapid detection and identification of mycobacterium directly in clinical samples but also of resistance genes for early diagnosis of MDR-TB and dealing with them quickly. However, the performance of these techniques does not sufficiently cover the diagnosis of smear-negative tuberculosis, extrapulmonary forms, children- and immune-compromised tuberculosis where sensitivity is limited. The diagnosis of latent tuberculosis is reinforced by the in vitro release testing of gamma interferon overcoming the lack of specificity of the tuberculin skin test. Despite considerable progress, more amelioration is still needed to improve these techniques in order to extend them to the paucibacillary tuberculosis and to facilitate their access to low-resource countries.

[Management of multidrug-resistant tuberculosis]. / Prise en charge de la tuberculose multirésistante.

The emergence of drug-resistant TB in many countries has become a major public health problem and an obstacle to effective tuberculosis control. Multidrug-resistant tuberculosis (MDR-TB), which is most often the result of poor adherence, is a particularly dangerous form of tuberculosis because it is caused by bacilli resistant to at least isoniazid and rifampicin, the two most effective anti-tuberculosis drugs. Techniques for rapid diagnosis of resistance have greatly improved the care of patients by allowing early treatment which remains complex and costly establishment, and requires skills and resources. The treatment is not standardized but it includes in all cases attack phase with five drugs (there must be an injectable agent and a fluoroquinolone that form the basis of the regimen) for eight months and a maintenance phase (without injectable agent) with a total duration of 20 months on average. Surgery may be beneficial as long as the lesions are localized and the patient has a good cardiorespiratory function. Evolution of MDR-TB treated is less favorable than tuberculosis with germ sensitive. The cure rate varies from 60 to 75% for MDR-TB, and drops to 30 to 40% for XDR-TB. Mortality remains high, ranging from 20 to 40% even up to 70-90% in people co-infected with HIV.

[Molecular comparison of Legionella pneumophila serogroup 1 isolated in Tunisia]. / Comparaison moléculaire de Legionella pneumophila sérogroupe 1 isolées en Tunisie.

Legionella pneumophila is a common cause of hospital and community-acquired pneumonia, being transmitted by inhalation of aqueous aerosols. Most outbreaks are linked to contaminated hot water systems and cooling towers. Our study was about the molecular typing of 35 strains of L. pneumophila including four clinical isolates and 31 environmental strains isolated from the distribution systems of 14 hotels. Among the clinical strains, two have the same pattern, however, all were different from the studied environmental strains. For the 31 environmental strains, ten patterns were obtained. Among which, a same pulsotype was found for four strains isolated from four different establishments. In addition, two different pulsotypes were found for strains isolated from the same establishment. The pulsed-field gel electrophoresis showed the existence of various patterns. Although cases of legionellosis were declared in these hotels, there are no epidemiological links between the clinical and environmental strains.