- Volume 66, Issue 7, 2017

Purpose. Staphylococcus epidermidis ATCC12228 lipoteichoic acid (LTA) inhibits TNFα production from keratinocytes that are activated with poly I:C. However, this effect has not been proven in clinical or commensal isolates.

Methodology. The <10 kDa fractions of S. epidermidis isolates from ocular infections (n=56), healthy skin (n=35) and healthy conjunctiva (n=32) were obtained. TNFα production was determined by elisa in HaCaT keratinocytes stimulated with poly I:C and with the <10 kDa fractions. LTA in the cytoplasmic membrane and in the <10 kDa fractions of the isolates was determined during bacterial growth by flow cytometry, Western blot and electrospray ionization mass spectrometry. The expression levels of ugtP, ltaA and ltaS were evaluated.

Results. Two populations of isolates were found: a population that inhibited TNFα production (TNFα-inhibitor isolates) and a population that did not inhibit it (TNFα non-inhibitor isolates). The cells from the TNFα-inhibitor isolates had less LTA in the cytoplasmic membrane compared to the cells from the TNFα non-inhibitor isolates (P<0.05). Similarly, LTA was detected in the supernatants of TNFα-inhibitor isolates, and it was absent in TNFα non-inhibitor isolates. High expression levels of the ugtP and ltaA genes in the 1850I (TNFα-inhibitor isolate) and 37HS (TNFα non-inhibitor isolate) isolates were found during bacterial growth. However, the ltaS gene had a low expression level (P<0.05) in the 37HS isolate.

Conclusion. The TNFα-inhibitor isolates release LTA due to high expression of the LTA synthesis genes. By contrast, TNFα non-inhibitor isolates do not release LTA due to low expression level of the ltaS gene.

Purpose. The occurrence of carbapenem- and colistin-resistance among Gram-negative bacteria is increasing worldwide. The aim of this study was to understand the distribution of carbapenem- and colistin-resistance in two areas in Tamil Nadu, India.

Methodology. The clinical isolates (n=89) used in this study were collected from two diagnostic centres in Tamil Nadu, India. The bacterial isolates were screened for meropenem- and colistin-resistance. Further, resistance genes bla NDM-1, bla OXA-48-like, bla IMP, bla VIM, bla KPC, mcr-1 and mcr-2 and integrons were studied. The synergistic effect of meropenem in combination with colistin was assessed.

Results. A total of 89 bacterial isolates were studied which included Escherichia coli (n=43), Klebsiella pneumoniae (n=18), Pseudomonas aeruginosa (n=10), Enterobacter cloacae (n=6), Acinetobacter baumannii (n=5), Klebsiella oxytoca (n=4), Proteus mirabilis (n=2) and Salmonella paratyphi (n=1). MIC testing showed that 58/89 (65 %) and 29/89 (32 %) isolates were resistant to meropenem and colistin, respectively, whereas 27/89 (30 %) isolates were resistant to both antibiotics. Escherichia coli, K. pneumoniae, K. oxytoca, Pseudomonas aeruginosa, and Enterobacter cloacae isolates were bla NDM-1-positive (n=20). Some strains of Escherichia coli, K. pneumoniae and K. oxytoca were bla OXA-181-positive (n=4). Class 1, 2 and 3 integrons were found in 24, 20 and 3 isolates, respectively. Nine NDM-1-positive Escherichia coli strains could transfer carbapenem resistance via plasmids to susceptible Escherichia coli AB1157. Meropenem and colistin showed synergy in 10/20 (50 %) isolates by 24 h time-kill studies.

Conclusion. Our results highlight the distribution of carbapenem- and colistin-resistance in Gram-negative bacteria isolated from the Tamil Nadu region in South India.

Purpose. Rapid and inexpensive tests for detecting extended-spectrum-β-lactamase (ESBL)-producing Enterobacteriaceae are needed, particularly in low-resource countries where infections with these bacteria constitute a major public health issue. The recently described ESBL NDP test performed well in developed countries. This study was designed to assess performance, cost and feasibility of this test in positive blood cultures, in Cotonou, Benin (West Africa).

Methodology. The test was performed on 175 positive Bactec broth blood cultures containing Enterobacteriaceae, and blindly compared with the double-disc synergy test (DDST) for the phenotypic detection of ESBL producers.

Results. There was a complete agreement between the ESBL NDP test and the DDST. On average, the time to give results was 37 min for a sample and the cost was US$ 7.3.

Conclusion. The ESBL NDP test is rapid, relatively affordable and performed well in our setting.

Purpose. The Limulus amebocytelysate (LAL) assay is widely used for the screening of lipopolysaccharide (LPS) in parenteral pharmaceuticals. However, correlation of LPS in Gram-negative bacterial infections by LAL assay has been problematic, partly due to the variable reactivity of different LPS structures. Recombinant factor C (rFC) has allowed the development of a new simple, specific and sensitive LPS detection system (PyroGene). In this work, the potential of the new assay for detecting various LPS structures has been investigated and compared with two LAL-based assays and a human monocyte activity assay.

Methodology. The activity of the various LPS structures has been investigated by PyroGene and two LAL-based assays and a human monocyte activity assay.

Results. The rFC assay detected most LPS structures in picogram quantities and the potency of E. coli, B. cepacia, Salmonella smooth and Salmonella R345 LPS was no different when measured with PyroGene or LAL assays. However, the reactivity of K. pneumoniae, S. marcescens, B. pertussis and P. aeruginosa LPS differed significantly between these assays. Importantly, pairwise correlation analysis revealed that only the PyroGene assay produced a significant positive correlation with the release of IL-6 from a monocytic cell line.

Conclusion. We conclude that the rFC-based assay is a good replacement for conventional LAL assays and as it correlates significantly with IL-6 produced by a human monocyte cell line it could potentially be more useful for detecting LPS in a clinical setting.

Purpose. The detection of galactomannan (GM) in bronchoalveolar lavage (BAL) fluid is an important surrogate marker for the early diagnosis and therapeutic monitoring of invasive aspergillosis (IA), regardless of the involved species of Aspergillus. Here, we utilized the Platelia Aspergillus GM enzyme immunoassay (Bio-Rad) to evaluate the GM index in BAL fluid samples from patients with proven, probable or putative IA due to Aspergillusflavus versus Aspergillusfumigatus.

Methodology. In a prospective study between 2009 and 2015, 116 BAL samples were collected from suspected IA patients referred to two university hospitals in Tehran, Iran.

Key findings. According to European Organization for Research and Treatment of Cancer and Mycoses Study Group and Blot criteria, 35 patients were classified as IA patients, of which 33 cases tested positive for GM above 0.5 and, among these patients, 22 had a GM index ≥1. Twenty-eight were culture positive for A. flavus and seven for A. fumigatus. The GM index for A. flavus cases was between 0.5–6.5 and those of A. fumigatus ranged from 1 to 6.5. The sensitivity and specificity of a GM index ≥0.5 in cases with A. flavus were 86 and 88 % and for A. fumigatus patients were 100 and 73 %, respectively.

Conclusion. Overall, the mean GM index in patients with A. fumigatus (3.1) was significantly higher than those of A. flavus (1.6; P-value=0.031) and the sensitivity of GM lower for A. flavus when compared to A. fumigatus. This finding has implications for diagnosis in hospitals and countries with a high proportion of A. flavus infections.

Background. Biofilm produced from Pseudomonas aeruginosa is the cause of infection induced by contact lenses, trauma and post-surgery infection. The aim of this study was to evaluate biofilm formation and the presence of the genes ndvB and tssC1 in ocular infection isolates of P. aeruginosa.

Methods. A total of 92 P . aeruginosa strains were collected from patients with ocular infection referred to Farabi Hospital between March 2014 and July 2015. Antibiotic susceptibility patterns were evaluated by the agar disc-diffusion method according to CLSI guidelines. PCR assays were used to detect ndvB and tssC1, genes associated with resistance in biofilm-producing P. aeruginosa isolates. Biofilm formation ability was examined by crystal violet microtitre plate assay.

Results. During the period of study, 92 P . aeruginosa were isolated from ocular infections including keratitis (n=84) and endophthalmitis (n=8). The highest resistance rates were seen against colistin (57.6 %) and gentamicin (50 %) and the lowest resistance rates were seen against imipenem (3.3 %), aztreonam (4.3 %), piperacillin-tazobactam (4.3 %), ceftazidime (4.3 %) and ciprofloxacin (5.4 %). Biofilm production ability was found in 100 % of the isolates. PCR assays showed that of the 92 P. aeruginosa isolates, 96.7 and 90.2 % harboured the genes ndvB and tssC1, respectively.

Conclusions. Our results showed a considerable ability of biofilm production, as well as the occurrence of biofilm-specific antimicrobial resistance genes (ndvB and tssC1), in P. aeruginosa isolates from ocular infections in Farabi Hospital.

Purpose. XpertMycobacterium tuberculosis/rifampicin (Xpert MTB/RIF) assay has been endorsed by the World Health Organization (WHO) for diagnosis of extrapulmonary tuberculosis (EPTB), while the sensitivity and specificity have not been fully evaluated. We aimed to evaluate the performance of Xpert MTB/RIF assay in the detection of different extrapulmonary specimens.

Methods. A total of 420 nonrespiratory specimens were detected with acid-fast bacilli (AFB) smear microscopy, solid culture, conventional drug susceptibility testing (DST) and Xpert MTB/RIF assay. Using solid culture and conventional DST as the gold standard, we assessed the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of the Xpert MTB/RIF assay for detecting MTB and rifampin resistance, respectively.

Results. When setting the solid culture results as the gold standard, the sensitivity, specificity, PPV, NPV and Kappa value of Xpert MTB/RIF assay and AFB smear results were 70.6 % (48/68), 91.96 % (318/346), 63.2 % (48/76), 94.1 % (318/338), 0.60, respectively. In addition, when compared with conventional DST results, the sensitivity, specificity, PPV, NPV and Kappa of the Xpert MTB/RIF assay for detecting rifampin resistance were 91.7 % (11/12), 100.0 % (47/47), 100.0 % (11/11), 97.9 % (47/48) and 0.95, respectively.

Conclusion. Compared with AFB smear and solid culture, Xpert MTB/RIF assay has high sensitivities and short detection time, so could be used as an alternative for the rapid diagnosis of EPTB and rifampin resistance in clinical practice.

The objective of the present study was to determine the frequency and age distribution of different Chlamydia trachomatis (CT) genotypes causing ophthalmia neonatorum (ON) in Hungary. Using CT specific PCR, we tested 76 conjunctival samples from symptomatic infants up to 3 months old in the National Centre for Epidemiology, Budapest between 2008 and 2016. CT tested positive in 30 of 76 conjunctival samples (39.5 %). The sequencing of the positive samples was successful in every case but one, and resulted in 48 % dominance for genotype E (14/29), followed by 24 % for genotype G (7/29), 10 % for J (3/29), 6.9 % for K and F (2/29), and 3.4 % for H (1/29). CT must still be regarded as a common pathogen causing ON in Hungary. Routine screening and treatment of pregnant women can be recommended to prevent these conditions. Chronic ON cases can be reduced by early diagnosis. Further research is needed to explain the dominance of genotypes E and G.

Purpose. In the last few decades, increasing microbial resistance to common antibiotics has attracted researchers' attention to the development of new classes of antibiotics such as antimicrobial peptides. Accordingly, the aim of the current study was to evaluate antimicrobial effects of the CM11 peptide alone and combined with common antibiotics against drug-resistant isolates of Brucella melitensis.

Methodology. A total of 50 pathogenic samples of B. melitensis were isolated from patients and their antibiotic susceptibility pattern was evaluated by E-test. Then, the synergistic reaction of the peptide with selected antibiotics was evaluated using a chequerboard procedure.

Results. Based on the susceptibility pattern of isolates, ciprofloxacin, rifampin, streptomycin and co-trimoxazole were used for synergistic study. According to the results, synergic effect was observed for streptomycin and co-trimoxazole in combination with the peptide while ciprofloxacin and rifampin showed partial synergy and additive effect, respectively. Consistent with these results, in the time-killing assay, a decrease in colony counts for streptomycin-peptide and co-trimoxazole-peptide was >2 Log10 while for ciprofloxacin-peptide and rifampin-peptide it was about 1.5 Log10 and <2 Log10, which represents synergy, partial synergy and additive interaction, respectively.

Conclusion. These results showed that by antibiotic-CM11 combination, their effective dose can be reduced particularly for drug-resistant isolates. In conclusion, considering the importance of brucellosis caused by B. melitensis in the Middle East beside reports on antibiotic resistance strains, especially against rifampin, which may literally lead to an increase in resistant strains of Mycobacterium tuberculosis in endemic areas, our findings can be used to develop a suitable alternative treatment for brucellosis, and with less risk.

Purpose. Urinary tract infection (UTI) can be community-acquired (Com-UTI) or catheter-associated (CAUTI) and may be associated with biofilm-producing organisms. A comparative analysis of biofilm-producing property (BPP), antibiotic-sensitivity and multi-drug resistance (MDR) and their relation with the BPP of isolates from Com-UTI and CAUTI has not yet been performed and necessitated this study.

Methodology. Objectives: (1) isolation of bacteria from CAUTI and Com-UTI and identification of their BPP, antibiotic-sensitivity and MDR status; (2) comparison of the isolates from CAUTI and Com-UTI as regards BPP, MDR status and their relation with BPP. Method: isolates from 100 cases each of Com-UTI and CAUTI were subjected to Congo redagar (CRA) and Safranin tube tests. Antibiotic susceptibility was investigated using the disc diffusion method. Both groups were compared regarding BPP, drug sensitivity and MDR status. Statistical analyses were performed using χ2 and Fisher’s exact tests.

Results. 76.19 % of isolates from Com-UTI and 60.72 % from CAUTI had BPP (P=0.0252; significant). The Safranin tube test detected more isolates with BPP than the CRA test. MDR is greater in CAUTI than Com-UTI (83.33 % versus 64.76 %; P=0.0039; significant). MDR is greater in isolates with BPP in both Com-UTI and CAUTI (76.47 and 62.35 %; non-significant).

Conclusions. BPP was found in both Com-UTI and CAUTI. When used together, the Safranin tube test and the CRA test increased the sensitivity of detecting BPP. MDR was higher in CAUTI than Com-UTI. MDR and BPP are not interrelated or associated, especially in settings where it is not certain that isolates were obtained from a well-formed biofilm. However, this does not rule out a higher incidence or prevalence of MDR in isolates with BPP taken directly from the biofilms.

Purpose. Molecular epidemiological investigations of the highly clonal Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) are important in outbreak detection and in tracking disease transmission. In this study, we developed and evaluated a multiple-locus variable-number tandem-repeats (VNTR) analysis (MLVA) assay for characterization of S. Typhi isolates from sub-Saharan Africa.

Methodology. Twelve previously reported VNTR loci were evaluated and an MLVA assay consisting of five polymorphic loci was adopted. The MLVA assay was developed for use on capillary electrophoresis systems by testing a collection of 50 S. Typhi isolates. This S. Typhi strain panel consisted of six outbreak related isolates and 44 epidemiologically unlinked isolates. Amongst these were nine S.Typhi haplotype H58 isolates.

Results. The MLVA assay characterized the 50 isolates into 47 MLVA profiles while PFGE analysis of the same isolates revealed 34 pulsotypes. MLVA displayed higher discriminatory power (Simpson’s index of diversity (DI) 0.998 [95 % confidence interval (CI) 0.995–1.000)] as compared to pulsed-field gel electrophoresis [Simpson’s DI 0.984 (95 % CI 0.974–0.994)].

Conclusion. The MLVA assay presented in this study is a simple, rapid and more accessible tool that serves as a good alternative to other molecular subtyping methods for S. Typhi.

Purpose. The aim of this study was to develop an immunogenic protective product against Shigella flexneri by employing a simple and safe heat treatment-based strategy.

Methodology. The physicochemical characteristics of naturally produced (OMV) and heat-induced (HT) outer-membrane vesicles from S. flexneri were examined, including a comparison of the protein content of the products. Toxicological and biodistribution studies, and a preliminary experiment to examine the protective effectiveness of HT in a murine model of S. flexneri infection, were also included.

Results. This method simultaneously achieves complete bacterial inactivation and the production of the HT vaccine product, leading to a safe working process. The obtained HT complex presented a similar morphology (electron microscopy) and chemical composition to the classical OMV, although it was enriched in some immunogens, such as lipoproteins, OmpA or OmpC, among others. The HT formulation was not toxic and biodistribution studies performed in mice demonstrated that the vaccine product remained in the small intestine after nasal administration. Finally, a single dose of HT administered nasally was able to protect mice against S. flexneri 2a.

Conclusion. The convenient and safe manufacturing process, and the preliminary biological evaluation, support the use of the self-adjuvanted HT complex as a new vaccine candidate to face shigellosis. Further development is required, such as additional immune analyses, to evaluate whether this new subunit vaccine can be useful in achieving full protection against Shigella.

This study aimed to understand the impact on virulence and fitness of mutations in specific genes found after adaptation of Klebsiella pneumoniae to colistin. Isolates with an increase in their inhibitory concentration (MIC) to colistin of 32- to >128-fold were shown to have mutations in mgrB, phoPQ and pmrAB, all known regulators of pathways affecting membrane lipid content. When these strains were used in studies in Galleria mellonella there was no clear correlation between mutations in specific genes per se and loss of virulence. Strains which showed sequence duplication in the HAMP-domain of PmrB showed reduced virulence but strains with point mutations in pmrAB showed no decrease in virulence. Similarly, specific mutations in mgrB in individual strains showed either loss of virulence or no effect/increased virulence. This study suggests that the impact on virulence may be independent of the colistin resistance mechanism and reflects differences in individual strain backgrounds.

Purpose. The purpose of the present study was to obtain a better understanding of the relationship between cell surface physiology and outer cellular envelope permeability for hydrophobic substances in mucoid and non-mucoid B. multivorans strains, as well as in two capsule-deficient derivatives of a mucoid parental strain.

Methodology. Cell surface hydrophobicity properties were determined using the hydrocarbon adherence method, while outer cell envelope accessibility and permeability for non-polar compounds were measured using hydrophobic antimicrobial agent susceptibility and fluorescent probe assays. Extracellular polysaccharide (EPS) production was assessed by cultivating strains of disparate origin on yeast extract agar (YEA) containing different sugars, while the resultant colonial and cellular morphological parameters were assessed macro- and microscopically, respectively.

Results/Key findings. The cell surfaces of all the strains were hydrophilic, impermeable to mechanistically disparate hydrophobic antibacterial agents and inaccessible to the hydrophobic probe N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of basal YEA with eight different sugars enhanced macroscopic EPS expression for all but one non-mucoid strain, with mannose potentiating the greatest effect. Despite acquisition of the mucoid phenotype, non-mucoid strains remained non-capsulated and capsulation of a hyper-mucoid strain and its two non-mucoid derivative strains was unaffected, as judged by microscopic observation.

Conclusion. These data support the conclusion that EPS expression and the consistent mucoid phenotype are not necessarily associated with the ability of the outer cell surface to associate with non-polar substances or cellular capsulation.

Purpose. This paper aims to evaluate the antimicrobial resistance of Esherichia coli isolates from children under 5 years old, with and without diarrhoea, who were hospital outpatients in Culiacan, Sinaloa, Mexico. It also looks at the antimicrobial activity of fruit extracts against selected multidrug-resistant (MDR) E. coli strains.

Methodology. A total of 205 E. coli isolates from stool samples were collected from 94 children under 5 years old who were outpatients from two hospitals in the city of Culiacan, Sinaloa, Mexico, during the autumn/winter of 2003/04; their resistance profiles to 19 commercial antimicrobials were investigated using the Kirby–Bauer method. The antibacterial activities of extracts/fractions of fruits (i.e. uvalama, Vitex mollis; ayale, Crescentia alata; and arrayan, Psidium sartorianum) were evaluated using the broth microdilution method.

Results. All E. coli isolates were susceptible to amikacin, nitrofurantoin and meropenem, and approximately 96 % were resistant to at least one antimicrobial, especially carbenicillin (93.2 %), cefuroxime sodium (53.7 %), ampicillin (40 %) and trimethoprim/sulfamethoxazole (35.1 %). Likewise, the frequency of MDR strains (44.9 %) was high, and no significant association with diarrhoea symptoms was found. Remarkably, all fruit extracts/fractions showed antibacterial activity against some, but not all, MDR isolates. The lowest minimal inhibitory concentration values were for the hexane fraction of arrayan (0.25 mg ml−1).

Conclusion. A high number of antimicrobial-resistant E. coli (especially to β-lactams and sulfonamides) and MDR isolates were detected in children under 5 years old, irrespective of diarrhoea symptoms; this is novel information for Culiacan, Sinaloa, Mexico. Moreover, our results showed that the studied fruit extracts/fractions are potential alternative or complementary treatments for MDR E. coli strains.