Showing papers in "International Microbiology in 2016"

Antimicrobial activity of

Abstract: This study was conducted to identify and evaluate the antimicrobial activity of some Lactobacillus isolates of chicken origin. Among 90 isolates 14 Lactobacillus species were distinguished using MALDI-TOF mass spectrometry and 16S-ARDRA. The dominant species was L. salivarius (34.4%), followed by L. johnsonii (23.3%), L. crispatus (13.3%) and L. reuteri (11.1%). All lactobacilli were screened for antimicrobial activity against wild-type strains of Salmonella enterica, Escherichia coli, and Clostridium perfringens. Results from the agar slab method showed that all Lactobacillus isolates were able to produce active compounds on solid media with antagonistic properties against these pathogens. The highest sensitivity to lactobacilli was observed in C. perfringens strains, and the lowest in E. coli. Lactobacillus salivarius exhibited particularly strong antagonism towards all of the indicator bacteria. Strains of L. ingluviei and L. johnsonii and one strain of L. salivarius (10d) selectively inhibited the growth of C. perfringens. No antimicrobial activity of many Lactobacillus isolates was observed when cell-free culture supernatant was used in a well diffusion assay. All Lactobacillus isolates exhibited the ability to produce H2O2 and proved to be hydrophobic (excluding one of L. salivarius). [Int Microbiol 19(1):57-67 (2016)]Keywords: Lactobacillus spp. · avian lactobacilli · antimicrobial activity · gut health · poultry pathogens

Distribution of virulence genes involved in biofilm formation in multi-drug resistant Acinetobacter baumannii clinical isolates

Abstract: Acinetobacter baumannii is an opportunistic bacterial pathogen that is the major cause of hospital-acquired infections. It has been shown that A. baumannii with high biofilm formation increases the risk of acquiring infection. In this study, the prevalence of virulence genes involved in biofilm formation was determined in 225 A. baumannii clinical isolates from three hospitals in Thailand. Most of the isolates were multidrug-resistant A. baumannii strains (86.2%). Among all isolates, 76.9% (173/225) showed biofilm formation ability. The association between biofilm forming ability and gentamicin resistance was found. The presence of virulence genes, epsA, bap, ompA, bfmS and blaPER-1 genes, was investigated by PCR. The prevalence of ompA, bfmS, bap, blaPER-1 and epsA genes among the isolated strains was 84.4%, 84%, 48%, 30.2%, respectively. Biofilm formation related genes, ompA and bap were associated with multidrug-resistant A. baumannii strains. The result of this study revealed that a high prevalence of biofilm-forming phenotypes among A. baumannii strains obtained from different hospitals. Effective strategies to prevent infection due to A. baumannii that produce biofilms are therefore needed. [Int Microbiol 19(2):121-129 (2016)].

Monitoring of airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios

Abstract: The first part of this review (“Monitoring of airborne biological particles in outdoor atmosphere Part 1: Importance, variability and ratios”) describes the current knowledge on the major biological particles present in the air regarding their global distribution, concentrations, ratios and influence of meteorological factors in an attempt to provide a framework for monitoring their biodiversity and variability in such a singular environment as the atmosphere Viruses, bacteria, fungi, pollen and fragments thereof are the most abundant microscopic biological particles in the air outdoors Some of them can cause allergy and severe diseases in humans, other animals and plants, with the subsequent economic impact Despite the harsh conditions, they can be found from land and sea surfaces to beyond the troposphere and have been proposed to play a role also in weather conditions and climate change by acting as nucleation particles and inducing water vapour condensation In regards to their global distribution, marine environments act mostly as a source for bacteria while continents additionally provide fungal and pollen elements Within terrestrial environments, their abundances and diversity seem to be influenced by the land-use type (rural, urban, coastal) and their particularities Temporal variability has been observed for all these organisms, mostly triggered by global changes in temperature, relative humidity, et cetera Local fluctuations in meteorological factors may also result in pronounced changes in the airbiota Although biological particles can be transported several hundreds of meters from the original source, and even intercontinentally, the time and final distance travelled are strongly influenced by factors such as wind speed and direction [Int Microbiol 2016; 19(1):1-1 3]Keywords: airborne biological particles · airbiota · bioaerosols · meteorological factors · air-genome ratios

The use of high throughput DNA sequence analysis to assess the endophytic microbiome of date palm roots grown under different levels of salt stress

Abstract: Date palms are able to grow under diverse abiotic stress conditions including in saline soils, where microbial communities may be help in the plant’s salinity tolerance. These communities able to produce specific growth promoting substances can enhance date palm growth in a saline environment. However, these communities are poorly defined. In the work reported here, the date palm endophytic bacterial and fungal communities were identified using the pyrosequencing method, and the microbial differential abundance in the root upon exposure to salinity stress was estimated. Approximately 150,061 reads were produced from the analysis of six ribosomal DNA libraries, which were prepared from endophytic microorganisms colonizing date palm root tissues. DNA sequence analysis of these libraries predicted the presence of a variety of bacterial and fungal endophytic species, some known and others unknown. The microbial community compositions of 30% and 8% of the bacterial and fungal species, respectively, were significantly (p ≤ 0.05) altered in response to salinity stress. Differential enrichment analysis showed that microbe diversity indicated by the Chao, Shannon and Simpson indices were slightly reduced, however, the overall microbial community structures were not significantly affected as a consequence of salinity. This may reflect a buffering effect by the host plant on the internal environments that these communities are colonizing. Some of the endophytes identified in this study were strains that were previously isolated from saline and marine environments. This suggests possible interactions with the plant that are favorable to salinity tolerance in date palm. [Int Microbiol 19(3):143-155 (2016)] Keywords: Phoenix dactylifera · endophytes · salt stress

Virulence factors and antimicrobial resistance in environmental strains of Vibrio alginolyticus.

Abstract: Vibrio alginolyticus has acquired increasing importance because this microorganism may be pathogenic to aquatic animals and humans It has been reported that some V alginolyticus strains carry virulence genes derived from pathogenic V cholerae and V parahaemolyticus strains In this work V alginolyticus was isolated from oyster samples acquired from a food-market in Mexico City Thirty isolates were identified as V alginolitycus Strains showed β-haemolysis and proteolytic activity and produced a capsule Strains displayed swimming and swarming motility and 933% of them produced siderophores Several genes encoding virulence factors were detected using PCR amplification These included proA, wza, vopD, vopB, hcp, vasH and vgrG genes, which were present in all strains Other genes had a variable representation: tdh (866%), lafA (966%), pvsA (62%) and pvuA (16%) The trh gene could not be amplified from any of the strains The antimicrobial resistance profile revealed that more than 90% of the strains were resistant to beta-lactams antibiotics, 60% to cephalotin, 45% to amikacin, 16% to cephotaxime, and 10% to pefloxacin, while 100% were susceptible to ceftriaxone The V alginolyticus strains isolated from oysters showed multiple resistance to antibiotics and several virulence factors described in well-characterized pathogenic vibrios [Int Microbiol 19(4):191-198 (2016)]

Monitoring of airborne biological particles in outdoor atmosphere. Part 2: Metagenomics applied to urban environments.

Abstract: The air we breathe contains microscopic biological particles such as viruses, bacteria, fungi and pollen, some of them with relevant clinic importance. These organisms and/or their propagules have been traditionally studied by different disciplines and diverse methodologies like culture and microscopy. These techniques require time, expertise and also have some important biases. As a consequence, our knowledge on the total diversity and the relationships between the different biological entities present in the air is far from being complete. Currently, metagenomics and next-generation sequencing (NGS) may resolve this shortage of information and have been recently applied to metropolitan areas. Although the procedures and methods are not totally standardized yet, the first studies from urban air samples confirm the previous results obtained by culture and microscopy regarding abundance and variation of these biological particles. However, DNA-sequence analyses call into question some preceding ideas and also provide new interesting insights into diversity and their spatial distribution inside the cities. Here, we review the procedures, results and perspectives of the recent works that apply NGS to study the main biological particles present in the air of urban environments. [Int Microbiol 19(2): 69-80 (2016)] Keywords: airborne biological particles · metagenomics · next-generation sequencing (NGS) · air biomonitoring · urban aerobiology

Antimicrobial activity of Lactobacillus strains of chicken origin against bacterial pathogenss.

Abstract: This study was conducted to identify and evaluate the antimicrobial activity of some Lactobacillus isolates of chicken origin. Among 90 isolates 14 Lactobacillus species were distinguished using MALDI-TOF mass spectrometry and 16S-ARDRA. The dominant species was L. salivarius (34.4%), followed by L. johnsonii (23.3%), L. crispatus (13.3%) and L. reuteri (11.1%). All lactobacilli were screened for antimicrobial activity against wild-type strains of Salmonella enterica, Escherichia coli, and Clostridium perfringens. Results from the agar slab method showed that all Lactobacillus isolates were able to produce active compounds on solid media with antagonistic properties against these pathogens. The highest sensitivity to lactobacilli was observed in C. perfringens strains, and the lowest in E. coli. Lactobacillus salivarius exhibited particularly strong antagonism towards all of the indicator bacteria. Strains of L. ingluviei and L. johnsonii and one strain of L. salivarius (10d) selectively inhibited the growth of C. perfringens. No antimicrobial activity of many Lactobacillus isolates was observed when cell-free culture supernatant was used in a well diffusion assay. All Lactobacillus isolates exhibited the ability to produce H2O2 and proved to be hydrophobic (excluding one of L. salivarius). [Int Microbiol 19(1):57-67 (2016)].

A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis.

Abstract: Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection. [Int Microbiol 2016; 49-55].

Lysozyme as a cotreatment during antibiotics use against vaginal infections: An in vitro study on Gardnerella vaginalis biofilm models.

Abstract: Bacterial vaginoses are frequent in women, most of them involving Gardnerella vaginalis. In more than 50% of the cases, usual antibiotic treatments are not capable of eliminating completely the infection, leading to recurrent vaginosis. In addition to the appearance of antibiotic resistance, recurrence can be due to the development of a biofilm by G. vaginalis. In vitro experiments on G. vaginalis biofilms showed that the biofilm protected bacteria from the antibiotic clindamycin. Also, recombinant human lysozyme (rhLys) was able to both degrade biofilms and prevent their formation. This degradation effect persisted whenever other vaginal commensal or pathogenic microorganisms were added to the culture and on each tested clinical biofilm-producing strain of G. vaginalis. The co-administration of rhLys and clindamycin or metronidazole improved both antibiotics' efficiency and lysozyme-driven biofilm degradation. The comparison of both clindamycin and metronidazole antibacterial spectra showed that metronidazole was preferable to treat vaginosis. This suggests that human lysozyme could be added as an anti-biofilm cotreatment to vaginal antibiotherapy, preferably metronidazole, against Gardnerella vaginalis infection in vivo. [Int Microbiol 19(2): 101-107 (2016)].

Investigation of twenty selected medicinal plants from Malaysia for anti-Chikungunya virus activity

Abstract: Chikungunya virus is a reemerging arbovirus transmitted mainly by Aedes mosquitoes. As there are no specific treatments available, Chikungunya virus infection is a significant public health problem. This study investigated 120 extracts from selected medicinal plants for anti-Chikungunya virus activity. The plant materials were subjected to sequential solvent extraction to obtain six different extracts for each plant. The cytotoxicity and antiviral activity of each extract were examined using African monkey kidney epithelial (Vero) cells. The ethanol, methanol and chloroform extracts of Tradescantia spathacea (Commelinaceae) leaves showed the strongest cytopathic effect inhibition on Vero cells, resulting in cell viabilities of 92.6% ± 1.0% (512 μg/ml), 91.5% ± 1.7% (512 μg/ml) and 88.8% ± 2.4% (80 μg/ml) respectively. However, quantitative RT-PCR analysis revealed that the chloroform extract of Rhapis excelsa (Arecaceae) leaves resulted in the highest percentage of reduction of viral load (98.1%), followed by the ethyl acetate extract of Vernonia amygdalina (Compositae) leaves (95.5%). The corresponding 50% effective concentrations (EC50) and selectivity indices for these two extracts were 29.9 ± 0.9 and 32.4 ± 1.3 μg/ml, and 5.4 and 5.1 respectively. Rhapis excelsa and Vernonia amygdalina could be sources of anti-Chikungunya virus agents. [Int Microbiol 19(3):175-182 (2016)]Keywords: Chikungunya virus · antivirals · cytotoxicity · sequential extraction · medicinal plants

Functional ecology of soil microbial communities along a glacier forefield in Tierra del Fuego (Chile).

Abstract: A previously established chronosequence from Pia Glacier forefield in Tierra del Fuego (Chile) containing soils of different ages (from bare soils to forest ones) is analyzed. We used this chronosequence as framework to postulate that microbial successional development would be accompanied by changes in functionality. To test this, the GeoChip functional microarray was used to identify diversity of genes involved in microbial carbon and nitrogen metabolism, as well as other genes related to microbial stress response and biotic interactions. Changes in putative functionality generally reflected succession-related taxonomic composition of soil microbiota. Major shifts in carbon fixation and catabolism were observed, as well as major changes in nitrogen metabolism. At initial microbial dominated succession stages, microorganisms could be mainly involved in pathways that help to increase nutrient availability, while more complex microbial transformations such as denitrification and methanogenesis, and later degradation of complex organic substrates, could be more prevalent at vegetated successional states. Shifts in virus populations broadly reflected changes in microbial diversity. Conversely, stress response pathways appeared relatively well conserved for communities along the entire chronosequence. We conclude that nutrient utilization is likely the major driver of microbial succession in these soils. [Int Microbiol 19(3):161-173 (2016)] Keywords: Functional genes · antibiotic resistance · GeoChip microarray · primary succession · chronosequence

Characterization of the microbiota associated to Pecten maximus gonads using 454-pyrosequencing

Abstract: A next-generation sequencing (NGS) approach was used to study the microbiota associated to Pecten maximus broodstock, applying pyrosequencing of PCR-amplified V1-V4 16S rRNA gene regions. We analysed the resident bacterial communities in female and male scallop gonads before and after spawning. DNA samples were amplified and quality-filtered reads were assigned to family and genus taxonomic levels using the Ribosomal Database Project classifier. A total of 18,520 sequences were detected, belonging to 13 phyla, including Proteobacteria (55%), Bacteroidetes (11,7%), Firmicutes (3%), Actinobacteria (2%) and Spirochaetes (1,2%), and 110 genera. The major fraction of the sequences detected corresponded to Proteobacteria, Beta- and Gammaprotebacteria being the most abundant classes. The microbiota of P. maximus gonad harbour a wide diversity, however differences on male and female samples were observed, where female gonad samples show a larger number of genera and families. The dominant bacterial genera appeared to be Delftia, Acinetobacter, Hydrotalea, Aquabacterium, Bacillus, Sediminibacterium, Sphingomonas, and Pseudomonas that were present among the four analysed samples. This next generation sequencing technique, applied for the first time in P. maximus (great scallop) gonads was useful for the study of the bacterial communities in this mollusc, unravelling the great bacterial diversity in its microbiota. [Int Microbiol 19(2): 93-99(2016)]Keywords: Pecten maximus · gonads microbiota · next-generation sequencing (NGS) · molluscs pathogens · aquaculture

Spatial homogeneity of bacterial and archaeal communities in the deep eastern Mediterranean Sea surface sediments.

Abstract: The diversity of microorganisms inhabiting the deep sea surface sediments was investigated in 9 stations (700-1900 m depth) in the Levantine basin by 454 massive tag sequencing of the 16S rDNA V4 region using universal primers. In total, 108,811 reads (an average of 10,088 per sample) were assigned to 5014 bacterial and 966 archaeal operational taxonomic units (OTUs; at 97% cut off). The 55% of the reads were of archaea, indicating dominance of archaea over bacteria at eight of the stations. The diversity and estimated richness values were high (e.g., H´ ranged from 5.66 to 7.41 for bacteria). The compositions of the microorganisms at all stations were remarkably similar, with Bray-Curtis similarities of 0.53–0.91 and 0.74–0.99 for bacterial and archaeal orders respectively. At two stations, very high abundances of only a few genera (Marinobacterium, Bacillus, Vibrio, Photobacterium) were accountable for the dissimilarities documented compared to the other deep sea stations. Half of the bacterial reads (51%) belonged to the phylum Proteobacteria, comprising mainly Gammaproteobacteria (41–72% of the proteobacterial reads per sample), Deltaproteobacteria (12–29%), Alphaproteobacteria (7–18%) and Betaproteobacteria (3–14%). The most abundant bacterial family was Sinobacteraceae (order Xanthomonadales) with 5–10% of total bacterial reads per sample. Most abundant reads (15.4% of all microbial reads) were affiliated with Marine Group 1 archaea, putatively capable of ammonia oxidation (213 OTUs), and bacteria involved in nitrification were found in all samples. The data point to the significant role that chemolithotrophic carbon assimilation and nitrification fill in the oligotrophic deep sea Levant sediments. [Int Microbiol 19(2): 109-119 (2016)] Keywords: deep sea sediments · eastern Mediterranean · microbial communities · ammonia oxidizing Archaea (AOA) · Israel

Phosphate: from stardust to eukaryotic cell cycle control.

Abstract: Phosphorus is a pivotal element in all biochemical systems: it serves to store metabolic energy as ATP, it forms the backbone of genetic material such as RNA and DNA, and it separates cells from the environment as phospholipids. In addition to this "big hits", phosphorus has recently been shown to play an important role in other important processes such as cell cycle regulation. In the present review, we briefly summarize the biological processes in which phosphorus is involved in the yeast Saccharomyces cerevisiae before discussing our latest findings on the role of this element in the regulation of DNA replication in this eukaryotic model organism. We describe both the role of phosphorus in the regulation of G1 progression by means of the Cyclin Dependent Kinase (CDK) Pho85 and the stabilization of the cyclin Cln3, as well as the role of other molecule composed of phosphorus-the polyphosphate-in cell cycle progression, dNTP synthesis, and genome stability. Given the eminent role played by phosphorus in life, we outline the future of phosphorus in the context of one of the main challenges in human health: cancer treatment. [Int Microbiol 19(3):133-141 (2016)].

Atypical enteropathogenic Escherichia coli (aEPEC) in children under five years old with diarrhea in Quito (Ecuador).

Abstract: Enteropathogenic Escherichia coli (EPEC) remain one the most important pathogens infecting children and they are one of the main causes of persistent diarrhea worldwide. In this study, we have isolated EPEC from 94 stool samples of children under five years old with diarrheal illness in the area of Quito (Ecuador), and we have determined the occurrence of the two subtypes of EPEC, typical EPEC (tEPEC) and atypical (aEPEC), by PCR amplification of the genes eae (attaching and effacing) and bfp (bundle- forming pilus). Typical EPEC is positive for eae and bfp genes while aEPEC is positive only for eae. Our results suggest that aEPEC is the most prevalent subtype in Quito (89.36 %), while subtype tEPEC is less prevalent (10.64 %). [Int Microbiol 19(3):157-160 (2016)].

Characterization of Escherichia coli clinical isolates causing urinary tract infections in the community of Chilpancingo, Mexico.

Abstract: The use of invasive techniques, such as intravascular catheter insertion, and the formation of biofilms in several devices by methicillin-resistant Staphylococcus aureus (MRSA) have contributed to the increased number of septic patients, morbidity and mortality This study aimed to evaluate the virulence of strains through catheter colonization and identification of microbial biofilm, as well as pathological changes on the colonized skin An experimental biofilm formation model utilized catheter fragments implanted subcutaneously in 25 Swiss mice The technique consisted of inoculating a catheter fragment on the back of each animal, followed by intradermal inoculation of 50 μl of bacterial suspension at 10 × 10⁷ colony forming units/ml After 96 h, catheters were removed for macroscopic analysis and evaluated through culture Local skin fragments were also extracted for histopathology analysis Staphylococcus aureus can adhere to catheters, colonize and form biofilms The high amount of viable bacterial cells colonizing catheters and virulence factors can lead to severe infections of skin and adjacent tissues [Int Microbiol 19(4): 199-207 (2016)]

Haloalkalitolerant Actinobacteria with capacity for anthracene degradation isolated from soils close to areas with oil activity in the State of Veracruz, Mexico.

Abstract: The use of native strains of microorganisms from soils is an excellent option for bioremediation To our knowledge, until now there has been no other group working on the isolation of Actinobacteria from contaminated soils in Mexico In this study, samples of soils close to areas with oil activity in the State of Veracruz, Mexico, were inoculated for the isolation of Actinobacteria The strains isolated were characterized morphologically, and the concentrations of NaCl and pH were determined for optimal growth Strain selection was performed by the detection of a phylogenetic marker for Actinobacteria located at the 23S rRNA gene, followed by species identification by sequencing the 16S rRNA gene Several haloalkalitolerant Actinobacteria were isolated and identified as: Kocuria rosea, K palustris, Microbacterium testaceum, Nocardia farcinica and Cellulomonas denverensis Except for C denverensis, the biomass of all strains increased in the presence of anthracene The strains capacity to metabolize anthracene (at 48 h), determined by fluorescence emission, was in the range of 46–54% During this time, dihydroxy aromatic compounds formed, characterized by attenuated total reflectance Fourier transform infrared spectroscopy bands of 1205 cm–1 and 1217 cm–1 Those Actinobacteria are potentially useful for the bioremediation of saline and alkaline environments contaminated with polycyclic aromatic hydrocarbon compounds [Int Microbiol 2016; 19(1):15-26]Keywords: Kocuria · Microbacterium · haloalkalitolerant Actinobacteria · anthracene degradation · State of Veracruz, Mexico

Antimicrobial activity of strains of chicken origin against bacterial pathogens

Abstract: This study was conducted to identify and evaluate the antimicrobial activity of some Lactobacillus isolates of chicken origin. Among 90 isolates 14 Lactobacillus species were distinguished using MALDI-TOF mass spectrometry and 16S-ARDRA. The dominant species was L. salivarius (34.4%), followed by L. johnsonii (23.3%), L. crispatus (13.3%) and L. reuteri (11.1%). All lactobacilli were screened for antimicrobial activity against wild-type strains of Salmonella enterica, Escherichia coli, and Clostridium perfringens. Results from the agar slab method showed that all Lactobacillus isolates were able to produce active compounds on solid media with antagonistic properties against these pathogens. The highest sensitivity to lactobacilli was observed in C. perfringens strains, and the lowest in E. coli. Lactobacillus salivarius exhibited particularly strong antagonism towards all of the indicator bacteria. Strains of L. ingluviei and L. johnsonii and one strain of L. salivarius (10d) selectively inhibited the growth of C. perfringens. No antimicrobial activity of many Lactobacillus isolates was observed when cell-free culture supernatant was used in a well diffusion assay. All Lactobacillus isolates exhibited the ability to produce H2O2 and proved to be hydrophobic (excluding one of L. salivarius). [Int Microbiol 19(1):57-67 (2016)] Keywords: Lactobacillus spp. · avian lactobacilli · antimicrobial activity · gut health · poultry pathogens

Prevalence of Campylobacter spp. in diarrhoea samples from patients in New South Wales, Australia.

Abstract: Campylobacteriosis is a leading cause of bacterial foodborne disease in many industrialized countries including Australia. New South Wales (NSW) is the most populous state in Australia yet the lack of any Campylobacter species surveillance programs has led to a knowledge gap in the importance of these pathogens as causes of diarrhoea. The data collected in this study demonstrated a need for such programs. In this study, 400 human clinical fecal samples were collected from two NSW locations, Western Sydney and Wagga Wagga, and tested for the presence of Campylobacter spp. Patients were clustered by location, age and gender to assess Campylobacter spp. prevalence within these groups between the two regions. The frequency of Campylobacter spp. was higher in males compared to females in the age groups 0-4 and 5-14 years; 6.4% and 1.0%, and 8.2% and none, respectively. A second peak was noted in elderly adults compared with those in younger age groups. Based on the findings of the quantitative PCR analysis it was estimated that the age-adjusted prevalence of Campylobacter spp. associated diarrhoea was 159 cases per 100,000 persons. [Int Microbiol 2016; 19(1):33-37].