Phylogenetic Aspects of Antibiotic Resistance and Biofilm Formation of P. aeruginosa Isolated from Clinical Samples.

Introduction: Biofilm production and drug resistance phenomenon play a critical role in P. aeruginosa infections. Several genes, including psl, pel, brlR, and mex, are involved in the phenomenon. The aim of this study was to find the relationship between the mentioned genes and the sources of P. aeruginosa infections. Materials and Methods: Fifty-nine P. aeruginosa isolates detected from clinical specimens were used to determine antibiotic susceptibility patterns, prevalence of the genes using PCR, biofilm formation, biofilm eradication concentration assay (MBEC), and epidemiological characteristics using pulsed-field gel electrophoresis (PFGE). Results: The results showed that 35.6% and 16.94% of all the samples were isolated from urine and wounds, 81.33% of the isolates were biofilm producers, 27.11% were multidrug-resistant (MDR), and 100% of the main biofilm former genes belonged to pslA. 94.91% of the isolates possessed brlR and mexA, and 91.5% of them expressed pslA. It was also indicated that neither ciprofloxacin nor imipenem could eradicate the formed biofilms. Moreover, we could identify 81.4% distinctive restriction profiles among the isolates, using an 80% similarity cutoff point; brlR and pel genes were significantly (P=0.032; P=0.044) related to phylogenetic pulsotypes. Comparison of the dendrogram in the isolates revealed that the detected isolates from urine were present in 12 different pulsotypes. Conclusion: It was found that there was a relationship between MDR, biofilm production, and brlR and pel genes among the isolates. It is distinguished there were similar genetic patterns between detected isolates from urine and could be concluded that the urinary tract played a critical role in maintaining and transferring biofilm drug-resistant genes of P. aeruginosa in clinical sites. The study highlights the importance of urine in distribution of clinical biofilm formation and drug-resistant P. aeruginosa isolates.

Immuno-reactivity evaluation of Mce-truncated subunit candidate vaccine against Mycobacterium avium subspecies paratuberculosis challenge in the goat models.

BACKGROUND: Detection of an appropriate antigen with high immunogenicity can be a big step in the production of an effective vaccine for control of Johne's disease (JD). The aim of this study was to evaluate the efficacy of Mce-truncated protein as a subunit vaccine candidate for the control of JD in experimentally challenged goats. MATERIALS AND METHODS: Six healthy goat kids were immunized with Mce-truncated protein, and two goats were kept as controls. All kids were twice challenged orally with live Mycobacterium avium subspecies paratuberculosis(MAP) strain and half the goats from both the categories were sacrificed at 7 and 10 months after start of challenge study. Culture of MAP was performed from all the necropsied tissues to determine the true JD infection status. RESULTS: Mce-truncated protein only reacted with pooled vaccinated goat sera in western-blot. A significant increase in humoral immune response against Mce protein was also observed in vaccinated goats. Compared to the control group, vaccinated goats gained higher body weights and none of them shed MAP or showed histopatological lesions or colonization of MAP in their necropsy tissues. CONCLUSIONS: The new Mce protein based vaccine provided significant immunity in goats as they could meet the challenge with live MAP bacilli. Although the vaccine used in this study showed the high potential as a new effective vaccine for the control of JD, further validation study is still required to successfully implement the vaccine for JD control program.

Application of Bayesian modeling for diagnostic assays of Mycobacterium avium subsp. paratuberculosis in sheep and goats flocks.

BACKGROUND: This study aimed to screen the sera of goats and sheep from flocks suspected of Mycobacterium avium subsp. paratuberculosis (MAP) infection by a newly standardized Mce-truncated ELISA (Mt-ELISA) kit for the detection of antibodies against MAP. Four diagnostic applied tests were evaluated including Indigenous plate-ELISA (IP-ELISA), Mt-ELISA, fecal Polymerase Chain Reaction (f-PCR) and fecal culture (FC). MATERIALS AND METHODS: Assuming the absence of a gold standard, latent-class models in a Bayesian framework were used to estimate the diagnostic accuracy of the four tests for MAP. RESULTS: Mt-ELISA had higher Sensitivity (Se) in sheep (posterior median: 0.68 (95% Probability Interval (PI): 0.43-0.95), while IP-ELISA recorded the highest Se in goats as 0.83 (95% PI, 0.61-0.97). The f-PCR Se estimate slightly differed between species [sheep 0.36 (0.19-0.58), goats 0.19 (0.08-0.35)], while the Se of FC was similar between species [sheep 0.29 (0.15-0.51), goats 0.27 (0.13-0.45)]. The specificity estimates for all tests were high, close to unity, and similar between species. CONCLUSION: Overall, the results showed that the Mt-ELISA method can be used for MAP detection in small ruminants' flocks.

Biotyping of isolates of Pseudomonas aeruginosa isolated from human infections by RAPD and ERIC-PCR.

Pseudomonas aeruginosa is a significant mortality factor due to nosocomial infections in humans. P. aeruginosa has been known with severe infections, high incidence, and multiple drug resistance. The present study aims to rapidly diagnose and biotype the isolates of P. aeruginosa isolated from human infections in Shiraz hospitals and health centers. Ninety six different isolates were collected from skin, urine, sputum, blood, wound, central vein blood, body fluids and burn wounds between January 2016 and February 2017. After phenotypic confirmation, isolates were examined by PCR for molecular confirmation. Ninety three isolates were verified as P. aeruginosa in molecular analysis. Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and Random Amplified Polymorphic DNA (RAPD) were done for 67 isolates. In ERIC-PCR, the patterns obtained included 2-11 bands. The RAPD patterns obtained with primers 272 and 208 consisted of 3-11 and 1-12 bands respectively. Based on dice similarity coefficient of greater than 80%, 38, 45 and 38 groups were identified in ERIC, RAPD 272 and RAPD 208 respectively. The results showed that the isolates of P. aeruginosa have a high polymorphism apparently because of the high genetic variation.

Novel recombinant Mce-truncated protein based ELISA for the diagnosis of Mycobacterium avium subsp. paratuberculosis infection in domestic livestock.

Johne's disease (JD) is an infectious wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP) in domestic livestock of every country that has been investigated. Controlling JD is problematic due to the lack of sensitive, specific, efficient, and cost-effective diagnostic tests. A major challenge in the development of diagnostics like ELISA is the selection of an ideal antigen/(s) that is pathogen-specific and allows sensitive recognition. Therefore, the purpose of this study was to identify and use Mce-truncated protein-based ELISA assay for the diagnosis of MAP infection with high sensitivity and specificity. In silico epitope prediction by epitope mapping throughout the whole length of MAP2191 protein revealed that C-terminal portion of this protein presented potential T- and B-cell epitopes. Therefore, a novel Mce-truncated protein encoded by the selected region of MAP2191 gene was expressed, purified with Ni-NTA gel matrix and confirmed by SDS PAGE and western blot. A profiling ELISA assay was developed to evaluate sera from MAP infected and non-infected ruminant species for antibodies against Mce-truncated protein to infer the immunogenicity of this protein in the host. Using this Mce protein-based ELISA, 251 goats, 53 sheep, 117 buffaloes, and 33 cattle serum samples were screened and 49.4, 51.0, 69.2, and 54.6% animals, respectively, were found positive. Comparing with i-ELISA, the new Mce-based ELISA kit showed a relatively higher specificity but suffered from slightly reduced sensitivity. Mce-based ELISA excluded apparently false positive results of i-ELISA. Mce protein was found to be antigenic and Mce-ELISA test could be employed as a diagnostic test for JD in domestic livestock in view of the a relatively higher specificity and accuracy. The antigenic potential of Mce antigen can also be exploited for the development of a new vaccine for the control of MAP infection.

Molecular typing of Staphylococcus aureus from different sources by RAPD-PCR analysis.

Staphylococcus aureus is an opportunistic bacterium which is carried as a normal flora organism but has a major role in the epidemiology and pathogenesis of different staphylococcal infections in humans and animals. Fifty S. aureus isolated from banknotes, foods, human infections and bovine mastitis were subjected to DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) analysis to examine their genotypic polymorphism and investigate the amount of genetic relatedness among these various isolates. At 100% RAPD profile similarity level, isolates were classified into four, five and seven groups of the same clone, according to the RAPDPCR with OLP6, OLP11 and OLP13 primers, respectively. Amplification of the isolates resulted in several polymorphic bands ranged from >50 to >1500 bp in size. Maximum number of bands was obtained by primer OLP13 which produced seven bands in bovine mastitis isolates. Most polymorphisms were observed in isolates of bovine mastitis and the lowest were associated with human infections isolates. There was no relationship between the RAPD patterns and the sources of isolates, except the three clusters which showed host specificity and only included the strains from the same sources. The results confirm the wide genotypic diversity of the studied S. aureus strains. RAPD-PCR technique can be a valuable tool for assessing the genetic relationship, detection of polymorphism in S. aureus and tracing the sources and management of S. aureus infections.

Mammalian cell entry operons; novel and major subset candidates for diagnostics with special reference to Mycobacterium avium subspecies paratuberculosis infection.

Mammalian cell entry (mce) genes are the components of the mce operon and play a vital role in the entry of Mycobacteria into the mammalian cell and their survival within phagocytes and epithelial cells. Mce operons are present in the DNA of Mycobacteria and translate proteins associated with the invasion and long-term existence of these pathogens in macrophages. The exact mechanism of action of mce genes and their functions are not clear yet. However, with the loss of these genes Mycobacteria lose their pathogenicity. Mycobacterium avium subspecies paratuberculosis (MAP), the etiological agent of Johne's disease, is the cause of chronic enteritis of animals and significantly affects economic impact on the livestock industry. Since MAP is not inactivated during pasteurization, human population is continuously at the risk of getting exposed to MAP infection through consumption of dairy products. There is need for new candidate genes and/or proteins for developing improved diagnostic assays for the diagnosis of MAP infection and for the control of disease. Increasing evidences showed that expression of mce genes is important for the virulence of MAP. Whole-genome DNA microarray representing MAP revealed that there are 14 large sequence polymorphisms with LSPP12 being the most widely conserved MAP-specific region that included a cluster of six homologs of mce-family involved in lipid metabolism. On the other hand, LSP11 comprising part of mce2 operon was absent in MAP isolates. This review summarizes the advancement of research on mce genes of Mycobacteria with special reference to the MAP infection.

Recombinant fusion protein of Heparin-Binding Hemagglutinin Adhesin and Fibronectin Attachment Protein (rHBHA-FAP) of Mycobacterium avium subsp. paratuberculosis elicits a strong gamma interferon response in peripheral blood mononuclear cell culture.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease in all ruminants worldwide. Economic problems in dairy cattle and sheep industries, public health concern, persistence of MAP in the environment and lack of effective vaccines mentioned necessity of research about various antigens to introduce as vaccine candidates. Based on MAP pathogenesis, it seems that research about the production of new recombinant proteins to stimulate cell-mediated immunity is helpful. This study describes successful expression and purification of a chimeric fusion protein which consists of Heparin-Binding Hemagglutinin Adhesin (HBHA) and high antigenic region of Fibronectin Attachment Protein (FAP-P). Triggered antigen-specific IFN-γ response of isolated PBMCs from immunized goats to rHBHA-FAP and all crude proteins of MAP (PPD), was measured by ELISA. RESULTS: Significant increases were observed in the IFN-γ production level of peripheral blood mononuclear cells (PBMCs) stimulated by constructed chimeric protein from rHBHA-FAP and PPD vaccinated goats. Antigen-specific gamma interferon (IFN-γ) secretion in positive group (immunized by PPD) against rHBHA-FAP and test group (immunized by rHBHA-FAP) against PPD, also statistically insignificant rises between stimulation with rHBHA-FAP and PPD, suggested the potential and specificity of our chimeric protein to stimulate cell mediated immunity against MAP. CONCLUSIONS: Collectively, these results demonstrate that rHBHA-FAP elicits a strong IFN-γ production in PBMC culture. Therefore, further studies of the present product as a candidate vaccine in naturally infected animals should be conducted, to analyze its potential.

Control of paratuberculosis: who, why and how. A review of 48 countries.

Paratuberculosis, a chronic disease affecting ruminant livestock, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It has direct and indirect economic costs, impacts animal welfare and arouses public health concerns. In a survey of 48 countries we found paratuberculosis to be very common in livestock. In about half the countries more than 20% of herds and flocks were infected with MAP. Most countries had large ruminant populations (millions), several types of farmed ruminants, multiple husbandry systems and tens of thousands of individual farms, creating challenges for disease control. In addition, numerous species of free-living wildlife were infected. Paratuberculosis was notifiable in most countries, but formal control programs were present in only 22 countries. Generally, these were the more highly developed countries with advanced veterinary services. Of the countries without a formal control program for paratuberculosis, 76% were in South and Central America, Asia and Africa while 20% were in Europe. Control programs were justified most commonly on animal health grounds, but protecting market access and public health were other factors. Prevalence reduction was the major objective in most countries, but Norway and Sweden aimed to eradicate the disease, so surveillance and response were their major objectives. Government funding was involved in about two thirds of countries, but operations tended to be funded by farmers and their organizations and not by government alone. The majority of countries (60%) had voluntary control programs. Generally, programs were supported by incentives for joining, financial compensation and/or penalties for non-participation. Performance indicators, structure, leadership, practices and tools used in control programs are also presented. Securing funding for long-term control activities was a widespread problem. Control programs were reported to be successful in 16 (73%) of the 22 countries. Recommendations are made for future control programs, including a primary goal of establishing an international code for paratuberculosis, leading to universal acknowledgment of the principles and methods of control in relation to endemic and transboundary disease. An holistic approach across all ruminant livestock industries and long-term commitment is required for control of paratuberculosis.

A Novel Approach to Deliver a Mycobacterium avium subsp. paratuberculosis Antigen in Eukaryotic Cells.

This study was aimed to express and deliver a Mycobacterium avium subsp. paratuberculosis antigen to macrophages using salmonella as carrier. The coding sequence of a fibronectin attachment protein which is expressed by Mycobacterium avium subsp. paratuberculosis was cloned into pcDNA3.1 (+) plasmid. The construct was introduced into the attenuated Salmonella typhimurium strain SL7207 (ΔhisG, ΔaroA) as carrier. In order to evaluate the delivery capacity of Salmonella and gene expression by antigen-presenting cells, the THP-1 derived macrophages were infected with the salmonella carrier. SDS-PAGE and western blot analysis showed the successful delivery and expression of targeted gene in THP-1 cell line. Although, in vitro stimulation of peripheral blood mononuclear cells with Salmonella containing plasmid did not trigger IFNγ production significantly. But it seems that this carrier can increase plasmid uptake and antigen expression by host intestinal antigen-presenting cells after mucosal administration. So, the construct can be used for further in vivo studies on the Salmonella carrier's efficiency in mycobacterial DNA vaccines.

Effectiveness of an inactivated paratuberculosis vaccine in Iranian sheep flocks using the Mycobacterium avium subsp paratuberculosis 316F strain.

BACKGROUND AND OBJECTIVES: Paratuberculosis (PTb) (John's disease) is an incurable chronic intestinal infection that mainly affects ruminants. PTb is caused by Mycobacterium avium subspecies paratuberculosis (MAP) with a global distribution. Despite evidences on MAP contribution in Crohn's disease its causal role is still a matter of controversy. In ruminant farming, vaccination is broadly accepted as an effective control measure of PTb. This article describes preparation and field trial of an inactivated PTb vaccine made from the MAP 316F strain. MATERIALS AND METHODS: Formulation of the vaccine was conducted based on the method traditionally used in the UK. Identity of the MAP strain was authenticated by PCR-IS900 and PCR-F57 tests. In the field, a group of 100 lambs (3-8 weeks old) were subcutaneously inoculated with the vaccine preparation under study. These animals, pre-vaccination, were all PTb ELISA negative. Serum level of antibody was determined by ELISA on days 0, 30, 60, 120 and 240, post-vaccination. RESULTS: In PCR-900 and PCR-F57, the MAP 316F strain produced two fragments of 560 and 704 bp length respectively, a confirmation of its identity as MAP bacterium. In the field trial and at the arranged time intervals, the achieved blood serum levels of antibody, attributable to the vaccine formulation, displayed considerably high values. CONCLUSION: Given that the PTb-caused economical losses in the Iranian environment are dramatically high and also the fact that future of state policy on control of PTb remains unknown, we belive vaccination of animals is the best recommendable practice.

Cloning and characterization of MAP2191 gene, a mammalian cell entry antigen of Mycobacterium avium subspecies paratuberculosis.

The aim of this study is to identify, clone and express a Mycobacterium avium subsp. paratuberculosis specific immunogenic antigen candidate, in order to develop better reagents for diagnosis and vaccines for the protection of the host. Therefore, MAP2191 gene (a member of MAPmce5 operon) from MAP, was isolated and characterized by Bioinformatics tools and in vitro experiments. Then, a novel Mce-whole protein encoded by MAP2191 gene was amplified and sub-cloned into E. coli. We tried to express the Mce/whole protein in different condition along with a positive expression control (pET28a-Mce/truncated plasmid that we know express well), to ensure that nothing is wrong regarding culture/induction condition. The level of the recombinant protein expression was analyzed by means of SDS-PAGE and Western blotting. Western blot analysis toward full-length MAP2191 protein and its truncation only demonstrated Mce/truncated protein. The concurrence of in-silico prediction of primary structure of MAP2191 protein results along with experimental results confirmed that expression of Mce/whole protein was affected by the hydrophobicity nature of this protein. Our data support the hypothesis that the presence of hydrophobic regions in protein structure can influence the level of recombinant protein expression. This stresses the importance of gene selection and the protein sequence checking of the hydrophobic content in any protein purification project in order to achieve a large amount of desirable proteins.

Epidemiology and molecular characteristics of methicillin-resistantStaphylococcus aureus from skin and soft tissue infections in Shiraz, Iran.

BACKGROUND/AIM: Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) infections are increasing in some regions of Iran. The aim of the current study was to assess the epidemiology and molecular characteristics of S. aureus isolated from patients with skin infections in Shiraz, Iran. MATERIALS AND METHODS: Swab samples were obtained from patients admitted to the skin and burn units of hospitals. The medical records of each patient were collected via questionnaire. All staphylococcal isolates were collected and examined by conventional methods for detecting S. aureus strains. PCR was used to detect S. aureus harboring the mecA and pvl genes. RESULTS: Out of 243 staphylococcal isolates, 55 (22.6%) S. aureus and 91 (37.4%) S. epidermidis were detected. Of the 45 patients, 21 (46.7%) were S. aureus carriers. The mecA gene was identified in 60% of S. aureus isolates, and the rest were sensitive to methicillin. Of the S. aureus isolates, 54.5% were positive for the pvl gene. CONCLUSION: This study revealed a high prevalence of PVL-positive MRSA strains in the evaluated hospitals. Thus, early diagnosis of infections caused by this pathogen seems to be necessary by intake screening allowing for proper treatment, especially in high-risk patients and in order to prevent the spread of infection.

Genotyping analysis of bovine, ovine, and caprine paratuberculosis in Iran: An IS900-RFLP study.

OBJECTIVE/BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) has been reported in Iranian cattle since 1965. Little is known on the population genetics of MAP in this Middle-Eastern State. A principle scope of this study was therefore genetic characterization of MAP isolates collected from farm animals in Iran. METHODS: Sixteen field isolates of MAP collected from bovine (n=8), ovine (n=3), and caprine (n=2) hosts in Alborz, Fars, Isfahan, Qazvin, Tehran, and Zanjan provinces were subcultured on mycobactin J-supplemented Herrold's egg slopes in order to provide the required genomic material. The laboratory strain MAP III & V was included as the reference strain. IS900-RFLP (Restriction Fragment length Polymorphism) was conducted using BsteII restriction enzyme. Application of IS900-RFLP genotyping on 16 Iranian MAP isolates in the present study classified them into six observable but similar types represented by two clustered and four orphan types. The laboratory strain MAP III & V displayed a totally different pattern easily distinguishable from that of Iranians. RESULTS: Detection of six genotypes among 16 wild isolates is an indication of a population with a potentially high level of diversity. We assume that, with inclusion of more field isolates, it is very likely that even higher diversity may be observed within the studied isolates. The different patterns displayed by the Iranians and the laboratory strain in this work might explain the independent evolutionary pathways these have gone through to evolve from their ancestral clones. CONCLUSION: Further description on population genetic of MAP in Iran urges for more epidemiological work using similar and alternative standard genotyping system.

Detection of Mycobacterium avium subspecies paratuberculosis infection in two different camel species by conventional and molecular techniques.

Paratuberculosis (John's disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat.

Association of Mycobacterium avium subspecies paratuberculosis infection with milk production and calving interval in Iranian Holsteins.

There are inconsistent results for the association of Mycobacterium avium subspecies paratuberculosis (MAP) infection with production and reproduction in dairy cows. Determination of these associations in each region is essential to encourage participation of dairy cattle producers in disease control programs. This study was conducted in Shiraz, southern Iran, to quantify the association of subclinical MAP infection with 305-day milk production and calving interval in Iranian Holsteins. A total of 21 dairy herds were selected for the study and in each herd, quarter milk samples were collected from ten to 12 dairy cows for PCR analysis. Data about parity, calving interval, length of lactation period, total milk production and 305-day milk production were also provided for each animal. Overall, 252 individual milk samples were collected. Herd- and individual-level prevalence of MAP infection were 23.8% (95% CI, 6.2-41.4%) and 3.2% (95% CI, 1.3-5.1%), respectively based on IS900 nested PCR. The results for 305-day milk production revealed a 248 kg reduction in positive cows compared with negative ones (P = 0.009). When cows from positive herds were compared with cows from negative herds, a 335-kg reduction in 305-day milk production (P = 0.005) and a 30-day increase in calving interval (P = 0.057) were observed in the former group. These findings support the previous results that paratuberculosis infection is negatively associated with the performance of the animals.

Risk factors for Mycobacterium avium subspecies paratuberculosis in Fars province (Southern Iran) dairy herds.

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level risk factors for infection with Mycobacterium avium subspecies paratuberculosis (MAP). Statistical analysis using multivariable logistic regression showed that contamination of udders of periparturient cows with manure (OR = 6.4, P = 0.02) and history of having suspected cases of Johne's disease in the herd (OR = 6.7, P = 0.04) were significantly associated with the herd infection status. No relationship between breed, herd size and other management practices with the infection status of the herd were found in this study. Implementing high sanitary measures in the farm, particularly with respect to manure handling and cleaning could be considered as one of the important aspects in controlling disease in the region as well as in the future educational effort.

Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds.

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection. Bulk-tank milk samples were collected from 110 dairy herds in the 3 districts (Shiraz, Marvdasht and Sepidan) of the province. Among study populations, 12 herds (11%, 95%CI: 5-17%) were positive for MAP infection based on IS900 nested PCR. The prevalence of positive milk samples in the three districts of Fars province was different ranging from 8.6% to 23% which was not statistically significant (P=0.19). It is recommended to conduct further epidemiologic studies to determine cow-level prevalence and risk factors for infection, and to evaluate the economic consequences of the MAP infection in the region.