Ectoparasites of dogs and cats in Albania

One hundred eighty-one dogs and 26 short-hair cats from suburban areas around Tirana, Albania were examined for ectoparasite infestation. The dogs were examined on several occasions from 2005 through 2009 representing three seasons: winter (December–February), spring (March–May), and summer (June–August); the cats were examined in late autumn (November). In addition, deep ear swab specimens of 30 dogs were examined for ear mites. The arthropod ectoparasite fauna of the dogs included two tick species (Rhipicephalus sanguineus and Ixodes ricinus), three mite species (Sarcoptes scabiei var. canis, Otodectes cynotis, and Demodex canis), three flea species (Ctenocephalides canis, Ctenocephalides felis, and Pulex irritans), and one louse species (Trichodectes canis). In the dogs, rates of infestation were 23.8% for R. sanguineus, 0.6% for I. ricinus, 4.4% for S. scabiei var. canis, 6.7% for O. cynotis, 0.6% for D. canis, 75.7% for C. canis, 5.0% for C. felis, 8.3% for P. irritans, and 6.6% for T. canis. Mixed infestation with two or three species of ectoparasites was recorded on 38.1% of the dogs. Fleas infested 75.7% dogs (geometric mean, 3.96; range, 1–80) and were observed in winter, spring, and summer with increasing prevalences of 64.3%, 75.9%, and 100%. Ticks parasitized 24.3% of the dogs (geometric mean, 0.41; range, 1–331). R. sanguineus ticks were recorded on 34.2% and 50% of the dogs examined in spring and summer, respectively, but were absent on the dogs during winter except for a single I. ricinus specimen observed. Prevalence of infestation with R. sanguineus, S. scabiei var. canis, C. felis, P. irritans, and T. canis did not differ between dogs ≤6 months and dogs >6 months of age; however, prevalence of infestation with C. canis was significantly (p?<?0.01) higher in dogs >6 months old. There was no difference between the sexes for the prevalences of infestation with those parasites. The examination of the cats revealed infestation with only one species of ectoparasite, C. felis (prevalence, 100%; geometric mean, 2.5; range, 1–9).     

Serodiagnostic evaluation of recombinant CdtB of <Emphasis Type="Italic">S</Emphasis>. Typhi as a potential candidate for acute typhoid

Typhoid fever caused by human restricted Salmonella typhi presents a considerable health burden on developing South-Asian nations like India. The suboptimal sensitivity and specificity associated with culture-based isolation of etiological agent and the extensively used surface antigen-based serological assays often lead to misdiagnosis and inappropriate antimicrobial treatment. The increasing reports of the emergence of resistant strains and undefined disease burden signify the critical need for an inexpensive, reliable, easy-to-use, and highly sensitive diagnostic test for typhoid fever. Utilizing S. typhi-specific and immunogenic antigens in sero-diagnostic assays could lead to precise diagnosis of acute typhoid and prompt treatment. In this study, we report cloning, expression, and purification of recombinant Cytolethal distending toxin subunit B (CdtB) of S. typhi, which is reported to be highly specific, immunogenic, and expressed only upon S. typhi infection. We further evaluated the purified recombinant CdtB for its diagnostic potential in an IgM-based indirect ELISA format using 33 human samples. Twenty-one serum samples from blood culture confirmed cases (n?=?21) of typhoid and 12 samples from healthy controls (n?=?12) were tested. The assay showed sensitivity of 100% and specificity of 83.3% respectively with positive and negative predictive values of 91.3 and 100% respectively. Efficient detection of specific IgM antibodies indicates that CdtB could be highly valuable in sero-diagnosis of acute typhoid and rapid screening of clinical samples.     

Molecular characterization of <Emphasis Type="Italic">Hepatozoon canis</Emphasis> from farm dogs in Pakistan

Hepatozoon canis is a tick-borne pathogen of canids, which is distributed worldwide. However, very little is known about this protozoan parasite in Pakistan. This study provides the first molecular evidence of H. canis from farm dogs from three agro-ecological zones of Punjab, Pakistan. A conventional PCR targeting the 18S rRNA gene was used to characterize H. canis from farm dogs from three districts, namely Kasur, Rawalpindi, and Muzaffargarh, in Punjab. Of 341 blood samples tested, 155 (45.5%) were positive for H. canis, 73 (61.3%) from Kasur, 46 (42.5%) from Rawalpindi, and 36 (31.5%) from Muzaffargarh. Phylogenetic analyses revealed that 18S rRNA sequences of H. canis from this study clustered in three clades with those of H. canis from previously published studies to the exclusion of all other Hepatozoon spp. included in the analysis. This study provides the first insight into H. canis from farm dogs in Pakistan. Furthermore, it lays a foundation for future studies of the parasite to assess the impact of canine hepatozoonosis in dogs from various agro-ecological zones in Pakistan where pet ownership of dogs is increasing.     

The importance of <Emphasis Type="Italic">Helicobacter pylori tnpA</Emphasis>, <Emphasis Type="Italic">tnpB</Emphasis>, and <Emphasis Type="Italic">cagA</Emphasis> genes in various gastrointestinal diseases

The cag (cytotoxin-associated gene) pathogenicity island (cagPAI) is one of the major virulence determinants of Helicobacter pylori (H. pylori). The purpose of this study was to investigate the association of the three genes (tnpA, tnpB, and cagA) in H. pylori isolated from Azerbaijani patients with the different gastrointestinal disease. A total of 362 gastric biopsies were collected from hospitals of Tabriz University of Medical Sciences, and were cultured on Brucella agar. The tnpA, tnpB, and cagA genes were detected by PCR. Of the total 264 H. pylori isolates, tnpA, tnpB, and cagA genes were detected in 120 (45.5%), 56 (21.2%) and 172 (65.2%), respectively. A significant association between tnpA and tnpB genes and clinical outcomes were found (P < 0.05). The cagA status was not related to clinical outcomes in our subjects. The predominant genotype among cag-PAI is the cagA. The prevalence of tnpA, tnpB, and cagA genes are high in patients with gastric cancer, and a significant association is revealed between tnpA and tnpB with gastric cancer.     

Strain variation and antigenic divergence among <Emphasis Type="Italic">Bordetella pertussis</Emphasis> circulating strains isolated from patients in Iran

Despite global efforts and widespread vaccination to control whooping cough (pertussis) caused by B. pertussis, the re-emergence of pertussis still is being reported all over the world. Antigenic divergence in B. pertussis virulence factors is one of the reasons of pertussis resurgence, resulting in dissimilarity of local and vaccine strains. In this study, clonal spread and variation of B. pertussis virulence factor in isolated strains from Iranian patients have been analyzed. A total of 100 B. pertussis isolates were obtained from Pertussis Reference Laboratory of Pasteur Institute of Iran. Real-time PCR were performed to confirm the B. pertussis strains. The genomic patterns of B. pertussis strains were analyzed by pulsed-field gel electrophoresis (PFGE). Predominant alleles of local strains were ptxP3, ptxA1, prn2, fim 2-1, fim3-2, and cya2. PFGE results showed 25 patterns clustered into 18 PFGE groups. A few similarities between the circulating isolates, vaccine, and standard strains were obtained. Significantly, 48% of the isolates showed dominant pattern with different allelic profiles from vaccine strains. According to the genomic profiles, the clonal spread was observed among the circulating strains. Predominant virulence factor profile was also comparable with other countries. It may be suggested that strain variation between vaccine and local strains may have an effect on pertussis resurgence in Iran like other parts of the world.     

<Emphasis Type="Italic">Annulotrema</Emphasis> (Monogenea: Dactylogyridae) from the gills of African tetras (Characiformes: Alestidae) in Lake Turkana,Kenya, with descriptions of four new species and a redescription of <Emphasis Type="Italic">A. elongata</Emphasis> Paperna and Thurston, 1969

Four new and four previously described species of Annulotrema were collected from the gills of four species (three genera, i.e. Alestes, Hydrocynus and Brycinus) of African tetras from Lake Turkana, Kenya: Annulotrema alestesnursi Paperna, 1973 from Brycinus nurse; Annulotrema ansatum n. sp., Annulotrema besalis ?ehulková, Musilová and Gelnar, 2014, Annulotrema bipatens n. sp., Annulotrema cucullatum n. sp., Annulotrema nili Paperna, 1973, and Annulotrema pontile n. sp. from Hydrocynus forskahlii; and Annulotrema elongata Paperna and Thurston, 1969 from Alestes baremoze and Alestes dentex. A. elongata is re-described on the basis of new material from A. baremoze. The sclerotized structures of the haptor and male copulatory organ of A. alestesnursi and A. elongata are illustrated from their type material. H. forskahlii is a new host record for A. besalis. The findings of A. besalis and A. elongata in Kenya represent a new locality records for these helminths. Three Annulotrema spp., namely A. besalis, A. elongata and A. pontile n. sp., share the same type of male copulatory organ, which may indicate a close relationship among these species.     

The relationship between polymorphism of four serotonic genes (<Emphasis Type="Italic">5-HTTL, 5-HT1A, 5-HT2A</Emphasis>, and <Emphasis Type="Italic">MAOA</Emphasis>) and personality traits in wrestlers and control group

The molecular genetic analysis of serotonin system genes (5-HTTL, 5-HT1A, 5-HT2A, and MAOA) in male and female wrestlers and in a control group has been performed. Comparative population genetics analysis of the 5-HTTL gene showed the highest frequency of the SS genotype 5-HTTLPR in athletes (p = 0.04), as well as a trend to higher frequencies of united genotypes of the locus 5-HTTLPR VNTR and SNP rs25531—S _A S _A (p = 0.06) compared to the control group. As for the markers of 5-HT1A (rs6295), 5-HT2A (rs6311), and MAOA (VNTR), we found no significant differences between the groups tested. Using an NEO PI-R questionnaire, possible correlations between the genotypes and psychological traits were analyzed in the studied samples. It was demonstrated that the athletic achievements of upper-tier athletes are associated with their lower openness to experience and higher conscientiousness. It was revealed that the factors of gender and 5-HT1A genotype should influence the self-rating for openness to experience. Furthermore, a combined effect of the level of sports achievements and 5-HT2A genotypes was found, as well as gender and 5-HT1A genotypes, on the self-rating for conscientiousness.     

Ecological features,pathogenic properties,and role of <Emphasis Type="Italic">Staphylococcus intermedius</Emphasis> group representatives in animal and human infectious pathology

The paper reports changes in the species nomenclature in the genus Staphylococcus that involve the most pathogenic representatives of the genus, the coagulase-positive staphylococci cluster. To date, this cluster includes six species in addition to Staphylococcus aureus: S. intermedius, S. schleiferi spp. coagulans, S. lutrae, S. hyicus, S. pseudintermedius, and S. delphini. Special attention was given to the representatives of the Staphylococcus intermedius group (SIG), which encompasses three closely related species, S. intermedius, S. pseudintermedius, and S. delphini, whose hosts are mammals and birds living in close proximity to human beings. The current data on the pathogenicity factors and role of SIG representatives in animal and human infectious pathologies were analyzed. The approaches to species identification, together with ecological and epidemiological features, and antibiotic susceptibility were considered. Specific biological features of S. pseudintermedius, the species most similar to S. aureus, are considered from the perspective of the properties of newly emerging pathogens.     

Telomere length in the gastric mucosa after <Emphasis Type="Italic">Helicobacter pylori</Emphasis> eradication and its potential role in the gastric carcinogenesis

The molecular mechanisms of gastric carcinogenesis after Helicobacter pylori (H. pylori) eradication remain unclear. We examined the telomere length of gastric mucosa samples after successful H. pylori eradication in patients without and those with gastric cancer. Telomere length was measured by the real-time PCR among four different groups of biopsies: gastric body from subjects without history of H. pylori infection (Hp-: n = 23), gastric body from cancer-free subjects after H. pylori eradication (cancer-free body: n = 24), gastric body from early gastric cancer patients diagnosed after H. pylori eradication (EGC body: n = 35) and its paired samples from adjacent mucosa of cancerous area (EGC ADJ: n = 35). The Hp-group presented the longest telomeres among the all groups (Hp- vs. all others, all P < 0.05). Samples from EGC body group showed shorter telomere length than the samples from cancer-free body groups (P < 0.05). Conversely, samples from EGC ADJ group showed rather longer telomere length compared to the EGC body group (P < 0.05), which was also confirmed by the comparison of 35 matched samples (P = 0.0007). Among the samples after H. pylori eradication, shorter telomere length was associated with higher expression of IL-1B and NF-kB (P < 0.0001, 0.0006, respectively). Longer telomere length was also associated with higher expression of TNF-A (P = 0.01). Telomere shortening seems to be important initial steps in gastric cancer predisposition after H. pylori eradication, while it might shift to lengthening to acquire more aggressive pathway to develop cancer.     

Morphological characterization and <Emphasis Type="Italic">HSP70</Emphasis>-, <Emphasis Type="Italic">IGS</Emphasis>-based phylogenetic analysis of two microsporidian parasites isolated from <Emphasis Type="Italic">Antheraea pernyi</Emphasis>

Two microsporidian isolates were extracted from single infected egg-laying tussah silk moth (Antheraea pernyi) in Liaoning Province, China. The microsporidia were subsequently grown in silk moth larvae, isolated, and subjected to morphological characterization (by light and transmission electron microscopy) and phylogenetic analysis (based on conserved genes). One type of spore was long-axis-oval in shape, measuring 4.71 × 1.95 μm, and the other type was short-axis-oval, measuring 3.64 × 2.17 μm. These dimensions were markedly different from those reported in the spores of the common microsporidia infecting A. pernyi, namely, Nosema pernyi (4.36 × 1.49 μm). A neighbor-joining phylogenetic tree based on HSP70 indicated that these microsporidia belonged to Nosema species and were closely related with Nosema bombycis and Nosema ceranae. Furthermore, in the phylogenetic tree based on the intergenic spacer (IGS) region, the long-axis-oval isolates were closely related and tended to form a clade away from the short-axis-oval isolates and N. pernyi isolates. The microsporidia isolated from A. pernyi clustered in one group. Nosema bombycis, Nosema spodopterae, and Endoreticulatus spp. appeared to be genetically distant from N. pernyi. The two isolates from A. pernyi fell in the Nosema group, but their spores differed from those of the spores of the common A. pernyi parasite N. pernyi, both in morphological and genetic aspects. The two isolates were designated Nosema sp. Ap (L) and Nosema sp. Ap (S). IGS was found to be informative in ascertaining phylogenetic relationships among species, and even closely related strains, of microsporidia.     

Association study of <Emphasis Type="Italic">Demodex</Emphasis> bacteria and facial dermatoses based on DGGE technique

The role of bacteria is unclear in the facial skin lesions caused by Demodex. To shed some light on this issue, we conducted a case-control study comparing cases with facial dermatoses with controls with healthy skin using denaturing gradient gel electrophoresis (DGGE) technique. The bacterial diversity, composition, and principal component were analyzed for Demodex bacteria and the matched facial skin bacteria. The result of mite examination showed that all 33 cases were infected with Demodex folliculorum (D. f), whereas 16 out of the 30 controls were infected with D. f, and the remaining 14 controls were infected with Demodex brevis (D. b). The diversity analysis showed that only evenness index presented statistical difference between mite bacteria and matched skin bacteria in the cases. The composition analysis showed that the DGGE bands of cases and controls were assigned to 12 taxa of 4 phyla, including Proteobacteria (39.37–52.78%), Firmicutes (2.7–26.77%), Actinobacteria (0–5.71%), and Bacteroidetes (0–2.08%). In cases, the proportion of Staphylococcus in Firmicutes was significantly higher than that in D. f controls and D. b controls, while the proportion of Sphingomonas in Proteobacteria was significantly lower than that in D. f controls. The between-group analysis (BGA) showed that all the banding patterns clustered into three groups, namely, D. f cases, D. f controls, and D. b controls. Our study suggests that the bacteria in Demodex should come from the matched facial skin bacteria. Proteobacteria and Firmicutes are the two main taxa. The increase of Staphylococcus and decrease of Sphingomonas might be associated with the development of facial dermatoses.     

Molecular characterization of <Emphasis Type="Italic">Giardia duodenalis</Emphasis> in dogs from Brazil

The intestinal protozoan parasite Giardia duodenalis (syn. Giardia intestinalis and Giardia lamblia) is a widespread enteric pathogen in human and domestic animals. This organism is one of the most common parasites in domestic dogs in Brazil. In this study, we determined the occurrence and genetic characterization of G. duodenalis isolated from dogs from south-central São Paulo state, Brazil. A total of 300 fecal samples were collected. Fecal specimens were screened for the presence of G. duodenalis using microscopy (zinc sulfate solution flotation technique) and polymerase chain reaction (PCR) targeting the small subunit ribosomal (SSU-rDNA) and glutamate dehydrogenase (GDH) genes. Genetic characterization was performed using restriction fragment length polymorphisms (RFLP) and sequencing analysis of the GDH gene. In addition, selected samples were further characterized by RFLP and sequencing of the β-giardin gene. The overall occurrence of G. duodenalis was 17.3% (52/300). The occurrence was higher in stray dogs (28%) than in household dogs (6.25%). Of the 36 PCR-positive samples that were selected for genotyping, only dog-specific genotype C (20 isolates), D (11 isolates) and mixed C?+?D (five isolates) isolates were detected in the study. This study provides current information on the infection rates of G. duodenalis genotypes in canine populations and describes for the first time the presence of mixed infections within host-specific C and D genotypes in dogs in Brazil. These genotypes were widespread and commonly found in domestic dogs living in urban and suburban environments of the studied area and confirmed the endemic status of Giardia in this region.     

Mammary candidiasis: molecular-based detection of <Emphasis Type="Italic">Candida</Emphasis> species in human milk samples

In this prospective and monocentric study, we investigated the performance of a commercialized real-time polymerase chain reaction (RT-PCR) test system for the specific detection of DNA from Candida albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis in human milk samples of patients suspicious of mammary candidiasis. For this purpose, 43 breast-feeding women with characteristic symptoms of mammary candidiasis and 40 asymptomatic controls were enrolled. By culture, Candida spp. were detected in 8.8 % (4/46) and 9.3 % (4/43) of patient and control samples, respectively. Candida albicans (2/46), C. parapsilosis (1/46), and C. guilliermondii (1/46) were present in patient samples, and C. lusitaniae (3/43) and C. guilliermondii (1/43) were present in the controls. After RT-PCR was applied, Candida spp. were found to be present in 67.4 % (31/46) and 79.1 % (34/43) of patient and control samples investigated, respectively. PCR detection of C. albicans and C. parapsilosis revealed only a low sensitivity and specificity of 67.4 % and 41.9 %, respectively. Our data do not support the use of Candida RT-PCR for sensitive and specific diagnosis of mammary candidiasis.     

Development of <Emphasis Type="Italic">T</Emphasis><Subscript><Emphasis Type="Italic">m</Emphasis></Subscript>-shift genotyping method for detection of cat-derived <Emphasis Type="Italic">Giardia lamblia</Emphasis>

To develop T _m -shift genotyping method for detection of cat-derived Giardia lamblia, two sets of primers with two GC-rich tails of unequal length attached to their 5′-end were designed according to two SNPs (BG434 and BG170) of β-giardin (bg) gene, and specific PCR products were identified by inspection of a melting curve on real-time PCR thermocycler. A series of experiments on the stability, sensitivity, and accuracy of T _m -shift method was tested, and clinical samples were also detected. The results showed that two sets of primers based on SNP could distinguish accurately between assemblages A and F. Coefficient of variation of T _m values of assemblage A and F was 0.14 and 0.07% in BG434 and 0.10 and 0.11% in BG170, respectively. The lowest detection concentration was 4.52 × 10^?5 and 4.88 × 10^?5 ng/μL samples of assemblage A and F standard plasmids. The T _m -shift genotyping results of ten DNA samples from the cat-derived G. lamblia were consistent with their known genotypes. The detection rate of clinical samples by T _m -shift was higher than that by microscopy, and their genotyping results were in complete accordance with sequencing results. It is concluded that the T _m -shift genotyping method is rapid, specific, and sensitive and may provide a new technological mean for molecular detection and epidemiological investigation of the cat-derived G. lamblia.     

Prevalence and molecular diversity of invasive <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> and <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> in a German tertiary care medical centre

Prevalence of invasive ß-haemolytic streptococci (BHS) at a tertiary care hospital and molecular diversity of S. pyogenes and S. dysgalactiae was studied. Between 2012 and 2016, all blood culture sets (n?=?55,839), CSF (n?=?8413) and soft tissue (n = 20,926) samples were analysed for BHS positivity using HYBASE software. Molecular profiles of 99 S. pyogenes and S. dysgalactiae were identified by sequencing of M protein genes (emm types) and multiplex PCR typing of 20 other virulence determinants. Streptococci contributed to 6.2% of blood, 10.7% of CSF and 14.5% of soft tissue isolates, being among the most common invasive isolates. The overall rates of invasive S. pyogenes, S. agalactiae, S. dysgalactiae and S. pneumoniae were 2.4, 4.4, 2.1, and 5.3%. Whereas S. pneumoniae was 1.5% more common in CSF samples, BHS isolates were 2-fold and 11-fold higher in bacteraemia and invasive soft tissue infections. Genetic BHS typing revealed wide molecular diversity of invasive and noninvasive group A and group G BHS, whereas one emm-type (stG62647.0) and no other virulence determinants except scpA were detected in invasive group C BHS. BHS were important invasive pathogens, outpacing S. pneumoniae in bacteraemia and invasive soft tissue infections. The incidence of S. dysgalactiae infections was comparable to that of S. pyogenes even with less diversity of molecular virulence. The results of this study emphasise the need for awareness of BHS invasiveness in humans and the need to develop BHS prevention strategies.     

Molecular epidemiology of <Emphasis Type="Italic">Treponema pallidum</Emphasis> in a Frontier region of the Russian Federation (Tuva Republic)

This article reports the results of typing 111 T. pallidum subsp. pallidum samples from Tuva Republic in 2013–2016. Samples were evaluated in molecular assays using variable genes arp, tpr (E, G, J), and tp0548 for the typing of Treponema pallidum strains. Seven molecular subtypes of the syphilis causative agent were identified over the whole period. The predominant type was 14 d/f (90.1%), followed by 14 b/f (1.8%), 14 c/f (1.8%), 14 d/g (0.9%) and 14 i/f (0.9%). Molecular subtypes 4 d/f and 9 d/f (0.9% each) identified in 2015, were previously described in Shanghai and northeastern China. In the period of 2015–2016 three cases of simultaneous presence of 2 arp types (9 and 14) in the clinical samples were identified for the first time. The comparison of the typing results with the current data of the syphilis molecular epidemiology allowed to conclude the similiarity of the T. pallidum subsp. population of the Tuva Republic with the Russian population in whole and its diversity from the populations described in the P. R. China and Western Europe.     

<Emphasis Type="Italic">Rickettsia</Emphasis> species in fleas collected from small mammals in Slovakia

Epidemiological and epizootiological studies of Rickettsia felis and other Rickettsia spp. are very important, because their natural cycle has not yet been established completely. In total, 315 fleas (Siphonaptera) of 11 species of Ceratophyllidae, Hystrichopsyllidae and Leptopsyllidae families were tested for the presence of Rickettsia species and Coxiella burnetii with conventional and specific quantitative real-time PCR assays. Fleas were collected from five rodent hosts (Myodes glareolus, Apodemus flavicollis, Apodemus agrarius, Microtus subterraneus, Microtus arvalis) and three shrew species (Sorex araneus, Neomys fodiens, Crocidura suaveolens) captured in Eastern and Southern Slovakia. Overall, Rickettsia spp. was found in 10.8 % (34/315) of the tested fleas of Ctenophthalmus agyrtes, Ctenophthalmus solutus, Ctenophthalmus uncinatus and Nosopsyllus fasciatus species. Infected fleas were coming from A. flavicollis, A. agrarius, and M. glareolus captured in Eastern Slovakia. C. burnetii was not found in any fleas. R. felis, Rickettsia helvetica, unidentified Rickettsia, and rickettsial endosymbionts were identified in fleas infesting small mammals in the Ko?ice region, Eastern Slovakia. This study is the first report of R. felis infection in C. solutus male flea collected from A. agrarius in Slovakia.     

Characterisation of VIM-2-producing <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> isolates from lower tract respiratory infections in a Spanish hospital

To analyse the antimicrobial phenotype, carbapenem mechanisms, integrons, virulence factors and molecular typing of 164 Pseudomonas aeruginosa isolates recovered from lower tract respiratory samples in a Spanish hospital (1 year) as well as the patients’ clinical data. Susceptibility testing to 12 antipseudomonal agents was determined by microdilution and metallo-beta-lactamase (MBL) phenotype by double disc method. The oprD gene was studied by PCR, sequencing and comparison with P. aeruginosa PAO1 sequence. Detection and characterisation of MBLs, class 1, 2 and 3 integrons, and virulence genes were studied by PCR and sequencing. The prevalence of carbapenem-resistant P. aeruginosa (CRPA) was 26.8%. MBL phenotype was detected in 52.3% CRPA, and all of them were disseminated throughout the intensive care unit. Most of the MBL-carrying patients presented respiratory disease, mechanical ventilation, tracheostomy, bacteraemia, ≥?30 hospitalisation days and previous treatment with carbapenems and/or ≥?3 different antimicrobial families. The bla_VIM-2 gene was the unique MBL encoding gene and was detected inside class 1 integrons. The class 1 integrons detected in 39 strains (23.8%) were associated with aminoglycosides (aadB, aadA1, aadA6, aacA4, aac(3)-I) and carbapenems resistance genes (bla_VIM-2). The aac(3)-I?+?aadA1 and bla_VIM-2 arrangements were the most prevalent ones. Thirty-one different PFGE patterns and 4 STs (ST175, ST235, ST253, ST973) were detected among the 39 intI1-positive isolates, being ST235 the most frequent. CRPA showed a great variety of alterations in oprD gene. The exoU⁺/exoS^? genotype was detected in 82.6% of bla_VIM-2-producing strains (ST235) and the exoU^?/exoS⁺ in the remaining 17.4% (ST973).     

New Mutations in Ciprofloxacin Resistant Strains of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolated from Guilan Province,Northern Iran

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a gram-negative bacterium that continues to be a major cause of opportunistic nosocomial infections. In P. aeruginosa, several fluoroquinolones resistance mechanisms have been proposed such as mutations in the target enzymes GyrA and ParC and upregulation of efflux pump MexAB-OprM. Fluoroquinolones inhibit the target enzymes DNA gyrase and topoisomerase IV, each comprised of two subunits: GyrA and GyrB, and ParC and ParE, respectively. In addition, fluoroquinolones were blocked by overexpression of MexAB-OprM induced through mutations in the regulatory genes mexR and nalC.

Materials and methods

In this study, 44 P. aeruginosa strains were isolated from different clinical samples of burn and infection from patients in some hospitals and laboratories in Guilan, which were identified by biochemical tests. The antibiotic resistance and susceptibility of strains was determined by Kirby Bauer method and microdilution method, and then PCR-sequencing was carried out to assess mutation in several genes involved in ciprofloxacin resistance.

Results

Of 44 isolates, 14 isolates were fluoroquinolone resistant. All 14 strains were nalidixic acid resistant with MIC = 1024 μg/mL. While ciprofloxacin resistance was showed in resistant isolates with MIC to be 32–1024 μg/mL. PCR-sequencing analysis showed that all 14 fluoroquinolone resistant isolates had one or two mutations in gyrA mutation. Mutations in parC, mexR and nalC were shown in some ciprofloxacin resistant isolates. Moreover, three strains had new mutation in mexR gene (111InsC and 262-263delAG).

Conclusions

It seems that high resistance to ciprofloxacin can simultaneously be the result of mutation in several genes such as topoisomerase enzymes and negative regulatory genes implicated in the expression of efflux pump systems in Guilan Province.