- Volume 68, Issue 12, 2019

Purpose. Carbapenemase-producing Enterobacteriaceae (CPE) have become a global concern and a serious threat to human health due to their resistance to multiple antibiotics. In this study, we identified and characterized CPE for the first time in Malawi, southeastern Africa.

Methodology. We investigated the possible presence of carbapenemases among a collection of 200 ceftriaxone-nonsusceptible Gram-negative clinical isolates obtained from five Malawian hospitals between January 2016 and December 2017, using both phenotypic and genotypic tests. Molecular typing of CPE was done by PFGE, multilocus sequence typing (ST) or phylogenetic grouping. Resistant plasmids were characterized by S1 PFGE, Southern blotting and conjugation assays.

Results. Out of 200 isolates, we detected 16 (8 %) CPE of which all originated from one referral hospital, Kamuzu Central Hospital, in the Central part of Malawi. Of 16 isolates, seven Klebsiella pneumoniae ST340/CC258 carried bla KPC-2, two Escherichia coli ST636 (phylogroup B2) carried bla NDM-5, six E. coli ST617 (phylogroup A) and one Klebsiella variicola carried bla OXA-48. All carbapenemases were plasmid-encoded, but only bla NDM-5-carrying plasmids could be conjugated. Most isolates co-harboured other β-lactamases and consequently exhibited a wider spectrum of resistance to commonly used antibiotics. We observed indistinguishable genetic profiles between strain types, despite originating from different wards, suggesting acquisition during admission and intra-hospital spread.

Conclusion. This report strongly suggests a probable existence of highly resistant various types of CPE organisms in Malawi including KPC-2-producing K. pneumoniae ST340/CC258, a known high-risk epidemic lineage.

Objective . Levonadifloxacin is a broad-spectrum anti-staphylococcal drug that is under development. We investigated the in vitro activity of levonadifloxacin against methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains phagocytized in THP-1 monocytes to evaluate its scope for treatment of intracellular staphylococcal infections.

Methods. The microdilution minimum inhibitory concentrations (MICs) of levonadifloxacin, moxifloxacin, levofloxacin and ciprofloxacin against MSSA ATCC 25923 and MRSA ATCC 43300 strains at pH 7.4±0.1 (original medium pH) and 5.5±0.1 (phagosome pH environment) were determined by following Clinical and Laboratory Standards Institute (CLSI) guidelines. The activity of antibiotics was investigated by extracellular and intracellular time–kill studies at 1–16× MIC concentrations. A suspension of ~5× log10 c.f.u. ml−1 test organism in supplemented RPMI 1640 medium was employed to determine the extracellular activity, while test organism phagocytized at a 4 : 1 ratio of bacteria to THP-1 monocytes was employed to investigate the intracellular activity. At intervals of 0, 2, 6 and 24 h, colony-forming unit (c.f.u.) counts were performed in triplicate on inoculated brain heart infusion (BHI) agar plates for both methods.

Results. At pH 7.4, the MIC of levonadifloxacin against both tested S. aureus strains was 2, 8 and 16 times lower than those of moxifloxacin, levofloxacin and ciprofloxacin, respectively. At pH 5.5, the MIC of levonadifloxacin was reduced by ≥8× against both tested S. aureus strains compared to its MIC at pH 7.4. In contrast, comparator quinolones showed a fourfold elevation in MIC at pH 5.5. In the study assessing the extracellular bactericidal effect, levonadifloxacin at 1× MIC manifested ≥4.5 log10 c.f.u. ml−1 killing for both S. aureus strains. Moxifloxacin and levofloxacin also showed bactericidal activity, while ciprofloxacin showed no killing. In intracellular conditions, levonadifloxacin manifested 1.0 log10 and 2.0 log10 killing for intracellular S. aureus ATCC 25923 and ATCC 43300, respectively. These killing effects were better overall than those of comparator quinolones.

Conclusions. Within a clinically achievable concentration range, levonadifloxacin achieved a 90–99 % intracellular reduction of MSSA and MRSA strains phagocytized in THP-1 monocytes. Therefore, levonadifloxacin has the potential to be a therapeutic option for the management of intracellular methicillin- and quinolone-resistant staphylococcal infections.

Introduction. Carbapenems are often described as the most effective weapon against infections caused by multidrug-resistant bacteria especially those belonging to the group of non-fermenting bacteria such as Pseudomonas . The main mechanisms leading to resistance are the hyperexpression of certain efflux pumps belonging to the resisto-nodular division and the lower expression of the transmembrane porin OprD, sometimes in combination with excessive production of the intrinsic AmpC. Carbapenemases are assumed to play a secondary role.

Aim. The aim of this study was to determine the exact mechanisms of carbapenem resistance in Pseudomonas aeruginosa isolates from the largest Bulgarian University hospital ‘St. George’- Plovdiv.

Methodology. A total of 32 clinical isolates collected from different patients’ samples resistant to imipenem and/or meropenem were examined via phenotypic and molecular-genetic tests.

Results. No metallo-enzyme production was detected. Three isolates were positive for OXA-50-encoding genes in two of them in combination with other oxacillinases or the bla VEB-1 gene. For the first time, OXA-50-producing P. aeruginosa have been reported in Bulgaria. The increased expression or hyperexpression of MexXY-OprM efflux pump was observed as the main mechanism of resistance. In most cases, it was combined with lower expression or lack of OprD with or without MexAB-OprM hyperexpression. No excessive production of AmpC was detected in comparison to the reference ATCC 27853 P . aeruginosa strain.

Conclusion. The increased expression or overexpression of MexXY-OprM efflux pumps is the leading cause of carbapenem resistance in our isolates Pseudomonas , detected in 94 % of the bacteria investigated.

Introduction. One of the most important resistant mechanisms in Gram-negative bacteria is extended spectrum β-lactamases (ESBLs). Harbour-related genes on plasmids, increase the risk of resistance transmission among commonly reported hospital infections.

Aim. This study was designed to explore the dissemination of Pseudomonas aeruginosa producing ESBLs on their plasmids recovered from the different wards of Amir-Al-Momenin burn center, Affiliated with Shiraz University of Medical Sciences.

Methodology. Among 256 isolates, 88 (34.38 %) P. aeruginosa strains were isolated from burn hospitalized patients. Samples were processed for antibiotic resistance using the Kirby–Bauer method while MIC was performed for colistin. MIC was used by the microdilution broth method as recommended by Clinical and Laboratory Standards Institute guidelines. Related studied genes were evaluated on extracted plasmids by the PCR method.

Results. According to the phenotypic and molecular steps, a total of 58 (65.91 %) and 74 (84.10 %) strains detected positive ESBLs, respectively. Based on antibiogram tests, a total of 63 (71.59 %) isolates were detected as multidrug resistant. All ESBL P. aeruginosa isolates showed identical antimicrobial susceptibility profiles. The genotypic prevalence of ESBLs for bla SHV, bla TEM, bla GES, bla OXA-10 and bla PSE genes was 47.73, 78.41, 5.58, 3.41, 4.55 %, respectively.

Conclusion. All P. aeruginosa strains producing ESBLs had plasmids containing related genes. The data indicated a high prevalence of ESBL among P. aeruginosa isolates in the southwest of the Iran burn center and their enzyme types were diverse.

Corynebacterium pseudotuberculosis is the causative agent of caseous lymphadenitis (CLA), a chronic disease of sheep and goats. Current methods for CLA diagnosis cannot identify all infected animals; therefore, the development of an improved diagnosis is essential. We evaluated recombinant phospholipase D (rPLD) protein individually or combined with rCP01850 or rCP09720 proteins for the detection of CLA in sheep. A total of 40 positive and 25 negative sera samples were analysed by ELISA using the recombinant proteins. ELISA using rPLD (E1), rPLD+rCP01850 (E2) and rPLD+rCP09720 (E3) showed 90, 92.5 and 97.5 % sensitivity and 92, 72 and 92 % specificity, respectively. The area under the receiver operating characteristic curves for E1, E2 and E3 was 0.925, 0.882 and 0.990, respectively. ELISA using rPLD +rCP09720 demonstrated the best sensitivity and specificity. Thus, the combination of these recombinant proteins in indirect ELISA has the potential for the diagnosis of CLA in sheep.

Introduction. Evidence for the clinical utility of bronchoalveolar lavage (BAL) galactomannan in the management of fungal disease outside of haemato-oncology patients is limited.

Aim. To determine how the introduction of BAL galactomannan testing impacted on the diagnosis and management of invasive aspergillosis and other fungal diseases in non-haemato-oncology patients.

Methodology. Retrospective review of all adult patients (age ≥16 years) without a diagnosis of haematological malignancy who had a positive BAL galactomannan from 1 November 2014 to 30 April 2018. Using electronic patient records we obtained demographic data, clinical details, laboratory investigations, relevant radiology and antimicrobial history for each case.

Results. In total, 121 episodes with a galactomannan OD index of ≥0.500 were included in the study; 29 cases (24 %) were felt to reflect fungal disease. Antifungal therapy was commenced as a direct consequence of a positive BAL galactomannan result in 13 patients where the ultimate diagnosis was subsequently considered to be non-mycological: associated medication-related side-effects in this group included deranged liver function tests (n=3), rash (n=1) and fever (n=1), related to amphotericin B (n=1) and voriconazole (n=4).

Conclusion. We show that vigilance is required when interpreting galactomannan results in non-haematology patients to avoid potentially harmful overtreatment.

Introduction. Colonization by Neisseria meningitidis is the pre-requisite for the development of disease. We present the findings of a cross-sectional investigation onto the oropharyngeal carriage of N. meningitidis and Neisseria species in the population aged 3 to 21 in Paraguay.

Aim. Carriage prevalence by age groups, risk factors associated with carriage, and phenotypic and genotypic characteristics of strains are described.

Methodology. We collected 2011 oropharyngeal swabs from consenting participants aged 3–21 years. Infants were recruited at immunization clinics, and older children and young adults were identified at schools and universities. A single oropharyngeal swab was collected and processed for the identification and isolation of Neisseria . Additionally, participants, or their legal guardian if these were minors, were requested to fill a standardized questionnaire.

Results. N. meningitidis was isolated in 42/2011 (2.1 %) participants, while other Neisseria spp. were identified in 306/2011 (15.2 %) subjects: N. cinerea and N. lactamica were identified in 39/2011 (1.9 %) and 43/2011 (2.2 %), respectively. Meningococcal strains belonged to ten different clonal complexes, of which six are associated with invasive disease (ST-32/ET5 complex, ST-11/ET37 complex, ST-103 complex, ST-167 complex, ST-35 complex and ST-41/44 complex/lineage 3).

Conclusion. Prevalence of N. meningitidis carriage was low compared to that reported from other settings, however, the overall carriage of Neisseria spp. (including N. meningitidis ) was comparable to meningococcal carriage prevalence reported in the literature. This study is the first of its kind conducted in Paraguay, and one of the few known in the Southern Cone of Latin America.

Introduction. Chronic pulmonary infection is associated with colonization with multiple micro-organisms but host–microbe and microbe–microbe interactions are poorly understood.

Aim. This study aims to investigate the differences in host responses to mono- and co-infection with S. aureus and B. cenocepacia in human airway epithelial cells.

Methodology. We assessed the effect of co-infection with B. cenocepacia and S. aureus on host signalling and inflammatory responses in the human airway epithelial cell line 16HBE, using ELISA and western blot analysis.

Results. The results show that B. cenocepacia activates MAPK and NF-κB signalling pathways, subsequently eliciting robust interleukin (IL)-8 production. However, when airway epithelial cells were co-treated with live B. cenocepacia bacteria and S. aureus supernatants (conditioned medium), the pro-inflammatory response was attenuated. This anti-inflammatory effect was widely exhibited in the S. aureus isolates tested and was mediated via reduced MAPK and NF-κB signalling, but not via IL-1 receptor or tumour necrosis factor receptor modulation. The staphylococcal effectors were characterized as small, heat-stable, non-proteinaceous and not cell wall-related factors.

Conclusion. This study demonstrates for the first time the host response in a S. aureus / B. cenocepacia co-infection model and provides insight into a staphylococcal immune evasion mechanism, as well as a therapeutic intervention for excessive inflammation.