ERIC-PCR Fingerprinting Research Articles

Antibiotyping, RAPD- and ERIC-PCR fingerprinting of Klebsiella pneumoniae clinical isolates at a tertiary reference hospital in Denpasar, Bali, Indonesia.

Klebsiella pneumoniae is a healthcare-associated infections agent and could be an extended spectrum β-lactamase (ESBL) producer. Understanding the transmission of this bacterium in a hospital setting needs accurate typing methods. An antibiogram is used to detect the resistance pattern of the isolates. Random Amplified Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR are rapid, technically simple, and easy-to-interpret DNA typing methods. This study aimed to evaluate the use of antibiotyping, RAPD-, and ERIC-PCR to investigate the heterogeneity of K. pneumoniae isolated from clinical specimens. The antibiograms of 46 K. pneumoniae clinical isolates were determined by Vitek® 2 Compact. All isolates underwent RAPD-PCR using AP4 primer and ERIC-PCR using ERIC-2 primer. The dendrogram was generated using the GelJ software and analyzed to determine its similarity. The analysis of antibiogram and the molecular typing diversity index was calculated using the formula of the Simpson's diversity index. About 71.7% of the isolates were ESBL-producers, and more than 80% of isolates were susceptible to amikacin, ertapenem, and meropenem. The antibiotyping produced 32 diverse types with DI = 0.964. In addition, the RAPD-PCR produced 47 different types with DI = 1, while ERIC-PCR was 46 (DI=0.999). Antibiotyping, RAPD- and ERIC-PCR showed powerful discrimination power among the isolates, supported the diversity of K. pneumoniae isolates in current study. These combination could be promising tools for clonal relationship determination, including in tracking the transmission of the outbreak's agent in hospital setting.

First Insights on Resistance and Virulence Potential of Escherichia coli from Captive Birds of Prey in Portugal.

Captive birds of prey are often used for pest control in urban areas, while also participating in falconry exhibitions. Traveling across the country, these birds may represent a public health concern as they can host pathogenic and zoonotic agents and share the same environment as humans and synanthropic species. In this work, Escherichia coli from the cloacal samples of 27 captive birds of prey were characterized to determine their pathogenic potential. Isolates were clustered through ERIC-PCR fingerprinting, and the phylogenetic groups were assessed using a quadruplex PCR method. Their virulence and resistance profile against nine antibiotics were determined, as well as the isolates' ability to produce extended-spectrum β-lactamases (ESBLs). The 84 original isolates were grouped into 33 clonal types, and it was observed that more than half of the studied isolates belonged to groups D and B2. Most isolates presented gelatinase activity (88%), almost half were able to produce biofilm (45%), and some were able to produce α-hemolysin (18%). The isolates presented high resistance rates towards piperacillin (42%), tetracycline (33%), and doxycycline (30%), and 6% of the isolates were able to produce ESBLs. The results confirm the importance of these birds as reservoirs of virulence and resistance determinants that can be disseminated between wildlife and humans, stressing the need for more studies focusing on these animals.

Isolation, Identification, and Characterization of Bacillus cereus Group Bacteria Isolated from the Dairy Farm Environment and Raw Milk in Tunisia.

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed β-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC, respectively. In addition, the detection rate of cytotoxin K (cytk), enterotoxin T (bceT), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

Molecular characterization of extended-spectrum β-lactamase-producing Escherichia coli isolated from lower respiratory tract samples between 2002 and 2019 in the Central Slovenia region

BackgroundAntibiotic resistance is one of the most serious global health problems and threatens the effective treatment of bacterial infections. Of greatest concern are infections caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC). The aim of our study was to evaluate the prevalence and molecular characteristics of ESBL-EC isolated over an 18-year pre-COVID period from lower respiratory tract (LRT) samples collected from selected Slovenian hospitals.Objectives and methodsAll isolates were identified by MALDI-TOF and phenotypically confirmed as ESBLs by a disk diffusion assay. Using a PCR approach, 487 non-repetitive isolates were assigned to phylogroups, sequence type groups, and clonal groups. Isolates were also screened for virulence-associated genes (VAGs) and antimicrobial resistance genes.ResultsThe prevalence of ESBL-EC isolates from LRT in a large university hospital was low (1.4%) in 2005 and increased to 10.8% by 2019. The resistance profile of 487 non-repetitive isolates included in the study showed a high frequency of group 1 blaCTX-M (77.4%; n = 377), blaTEM (54.4%; n = 265) and aac(6')-Ib-cr (52%; n = 253) genes and a low proportion of blaSHV and qnr genes. Isolates were predominantly assigned to phylogroup B2 (73.1%; n = 356), which was significantly associated with clonal group ST131. The ST131 group accounted for 67.6% (n = 329) of all isolates and had a higher number of virulence factor genes than the non-ST131 group. The virulence gene profile of ST131 was consistent with that of other extraintestinal pathogenic E. coli (ExPEC) strains and was significantly associated with ten of sixteen virulence factor genes tested. Using ERIC-PCR fingerprinting, isolates with the same ERIC-profile in samples from different patients, and at different locations and sampling dates were confirmed, indicating the presence of “hospital-adapted” strains.ConclusionOur results suggest that the ESBL-EC isolates from LRT do not represent a specific pathotype, but rather resemble other ExPEC isolates, and may be adapted to the hospital environment. To our knowledge, this is the first study of ESBL-EC isolated from LRT samples collected over a long period of time.

High resistance of fluoroquinolone and macrolide reported in avian pathogenic Escherichia coli isolates from the humid subtropical regions of Pakistan

ObjectivesThis study aimed to assess the antimicrobial resistance profile, virulence potential, and genetic characterization of avian pathogenic Escherichia coli (APEC) that cause colibacillosis in poultry. MethodsAntibiotic susceptibility testing (AST) was measured via the Kirby-Bauer disc diffusion method against 27 commonly used antibiotics. Phylogrouping, virulence-associated gene detection, and hybrid strain detection via multiplex polymerase chain reaction (PCR) and genetic diversity were analysed via ERIC-PCR fingertyping method. ResultsAST analysis showed 100% of isolates were multidrug-resistant (MDR) and highest resistance was against penicillin, tetracycline, and macrolide classes of antibiotics. The mcr-1 gene was present in 40% of the isolates, though only 4% of isolates were showing phenotypic resistance. Despite the scarce use of fluoroquinolone, carbapenem, and cephalosporin in the poultry sector, resistance was evident because of the high prevalence of extended-spectrum β-lactamase (ESBL) (53.7%) and other β-lactamases in APEC isolates. β-lactamase genotyping of APEC isolates revealed that 85.7% of isolates contained either blaCTX or blaTEM and around 38% of isolates were complement resistant. Growth in human urine was evident in 67.3% of isolates. Phylogroup B1 (51%) was the most prevalent group followed by phylogroups A (30.6%), D (13.61%), and B2 (4.76%). The most prevalent virulence-associated genes were fimH, iss, and tatT. Results showed that 26 isolates (17.69%) can be termed hybrid strains and APEC/EHEC (enterohemorrhagic E. coli) was the most prevalent hybrid E. coli pathotype. ERIC-PCR fingerprinting genotype analysis clustered APEC isolates in 40 groups (E1-E40). This study provides insights into the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. ConclusionsThe findings of this study provide insights into that the antibiotic resistance and virulence profiling of the APEC isolates in Pakistan. This data can inform future studies designed to better estimate the severity of the colibacillosis in poultry farms.

Virulence Characteristics, Antibiotic Resistance Patterns and Molecular Typing of Enteropathogenic Producing Escherichia coli (EPEC) Isolates in Eastern Province of Saudi Arabia: 2013-2014.

The Kingdom of Saudi Arabia has limited data on enteropathogenic Escherichia coli (EPEC). Therefore, this study was undertaken to contribute to EPEC surveillance and investigate the molecular epidemiology of EPEC strains that have been implicated in human infection in King Fahd Hospital of the University (KFHU) between 2013 and 2014 in the Eastern Province of Saudi Arabia. A total of 60 non-duplicate E. coli isolates associated with human gastroenteritis were included in this study. They were characterized using PCR to determine virulence genes, antimicrobial resistance patterns, and enterobacterial repetitive intergenic consensus (ERIC-PCR). Among the 60 strains, 20% of those examined were positive for the intimin eae and bfpA genes and identified as typical EPEC (tEPEC). Furthermore, 44 of E. coli strains tested positive for the eae gene only and revealed a high occurrence rate of 73.3% of atypical EPEC (aEPEC) within the overall examined strains. All strains were positive for the EAST1 gene, and none tested positive for the stx gene. More than 70% of EPEC strains were multi-drug resistant (MDR) and aEPEC strains with the highest proportion of this feature of MDR. ERIC-PCR fingerprint revealed a total of 19 ERIC types with eight related distinct clusters and a similarity rate cut-off with ≥90% homology from the identified isolates. A high antibiotic resistance rate was reported for first-line antibiotics, such as ampicillin, tetracycline, nalidixic acid, trimethoprim-sulfamethoxazole, noroxin, and ciprofloxacin.

Molecular Typing Reveals Environmental Dispersion of Antibiotic-Resistant Enterococci under Anthropogenic Pressure.

As a consequence of global demographic challenges, both the artificial and the natural environment are increasingly impacted by contaminants of emerging concern, such as bacterial pathogens and their antibiotic resistance genes (ARGs). The aim of this study was to determine the extent to which anthropogenic contamination contributes to the spread of antibiotic resistant enterococci in aquatic compartments and to explore genetic relationships among Enterococcus strains. Antimicrobial susceptibility testing (ampicillin, imipenem, norfloxacin, gentamycin, vancomycin, erythromycin, tetracycline, trimethoprim-sulfamethoxazole) of 574 isolates showed different rates of phenotypic resistance in bacteria from wastewaters (91.9–94.4%), hospital effluents (73.9%), surface waters (8.2–55.3%) and groundwater (35.1–59.1%). The level of multidrug resistance reached 44.6% in enterococci from hospital effluents. In all samples, except for hospital sewage, the predominant species were E. faecium and E. faecalis. In addition, E. avium, E. durans, E. gallinarum, E. aquimarinus and E. casseliflavus were identified. Enterococcus faecium strains carried the greatest variety of ARGs (blaTEM-1, aac(6′)-Ie-aph(2″), aac(6′)-Im, vanA, vanB, ermB, mefA, tetB, tetC, tetL, tetM, sul1), while E. avium displayed the highest ARG frequency. Molecular typing using the ERIC2 primer revealed substantial genetic heterogeneity, but also clusters of enterococci from different aquatic compartments. Enterococcal migration under anthropogenic pressure leads to the dispersion of clinically relevant strains into the natural environment and water resources. In conclusion, ERIC-PCR fingerprinting in conjunction with ARG profiling is a useful tool for the molecular typing of clinical and environmental Enterococcus species. These results underline the need of safeguarding water quality as a strategy to limit the expansion and progression of the impending antibiotic-resistance crisis.

Phylogenetic relationships among Bradyrhizobium species nodulating groundnut (Arachis hypogea L.), jack bean (Canavalia ensiformis L.) and soybean (Glycine max Merr.) in Eswatini

This study assessed the genetic diversity and phylogenetic relationships of rhizobial isolates obtained from root nodules of groundnut, jack bean and soybean planted in different locations within Eswatini. Seventy-six rhizobial isolates were studied using ERIC-PCR (enterobacterial repetitive intergenic consensus) fingerprinting and PCR amplification of 16S rRNA, housekeeping genes (atpD, dnaK, glnll and rpoB) and symbiotic genes (nifH and nodC). The dendrogram generated from the ERIC-PCR banding patterns grouped the test rhizobial isolates into 16 major clusters (Cluster I–XVI), with three isolates, namely TUTAHeS60, TUTGMeS3 and TUTAHeS127, forming outgroups of Clusters IV, VI and IX, respectively. Furthermore, the 76 test isolates were grouped into 56 ERIC-PCR types at 70% similarity level. The phylogenetic analysis of the 16S rRNA gene and multilocus sequence analysis of four housekeeping (atpD, dnaK, glnII and rpoB) and two symbiotic (nifH and nodC) genes showed that all three legumes (groundnut, jack bean and soybean) were nodulated by bacterial symbionts belonging to the genus Bradyrhizobium, with some isolates exhibiting high divergence from the known reference type strains. The results also showed that B. arachidis, B. iriomotense and B. canariense were the closest type strains to the groundnut isolates, while B. pachyrhizi and B. elkanii were the closest relatives to the bacterial symbionts associated with the nodulation of both jack bean and soybean. This study is the first report to describe of the bacterial symbionts nodulating jack bean in African soils.

Functional and genetic diversity of native rhizobial isolates nodulating cowpea (Vigna unguiculata L. Walp.) in Mozambican soils

Identification and symbiotic characterization of indigenous rhizobial isolates are the basis for inoculant formulations needed for sustainable grain legume production. This study screened for morpho-genetic diversity of indigenous cowpea nodulating rhizobia in farmers’ fields across two contrasting agroecological zones of Northern Mozambique. The photosynthetic function induced by the isolates in their homologous cowpea was assessed. The results showed high genetic variability among the isolates based on morphology and ERIC-PCR fingerprinting. The trap cowpea genotype did not influence the diversity of isolates collected from the two different agroecologies, suggesting that the cowpea-rhizobia compatibility may be conserved at species level. Phylogenetic analysis of the 16S rRNA gene assigned representative rhizobial isolates to species in the Bradyrhizobium and Rhizobium genera, with some isolates showing high divergence from the known reference type strains. The isolates from both agroecologies highly varied in the number and biomass of nodules induced in the homologous cowpea, resulting in variable plant growth and photosynthetic activities. A total of 72% and 83% of the isolates collected from the agroecological zones 7 and 8 were respectively classified as highly effective candidates with > 80% relative effectiveness compared to plants fertilized with nitrate, indicating that elite native strains populated the studied soils. Moreover, the top 25% of high N2-fixing isolates from the two agroecologies recorded relative effectiveness ranging from 115 to 154%, values higher than the effectiveness induced by the commercial Bradyrhizobium sp. strain CB756. These strains are considered as having potential for use in inoculant formulations. However, future studies should be done to assess the ecologically adaptive traits and symbiotic performance under field conditions.

Virotyping, genotyping, and molecular characterization of multidrug resistant Escherichia coli isolated from diarrheal patients of Bangladesh

Multidrug-resistant (MDR) Escherichia coli has been previously detected in other locations, but not in Chhatak, a diarrhea endemic region of Bangladesh. In this study, 36 E. coli isolated from diarrhea stools of adults and children from Chhatak were investigated for the prevalence of drug resistance, virulence and resistance related genes, genotyping, and other molecular characteristics. Antibiotic resistance profiling was performed by disk diffusion method whereas the presence of virulence genes, mobile genetic elements, plasmids, and resistance-related genes were monitored by PCR assay. Multiple resistance to drugs was observed in 94.4% of the isolates and 24 (66.7%) of the strains were found as enterotoxigenic E. coli (ETEC) pathogroup. Genotyping by ERIC-PCR fingerprinting revealed genetic heterogeneity among the MDR E. coli strains. Overall, 88% of E. coli strains possessed plasmids ranging from 0.6 to 140 MDa. Of the detected integrases (69%), intl1 was predominant (53%). Erythromycin esterase-B (ereB) was present in 8 out of 36 macrolide resistant strains (22%). Of 27 beta-lactam resistant isolates, 70%, 41%, and 33% carried blaCTX-M-1, blaTEM, and blaOXA, respectively. Of 23 tetracycline resistant strains, 13 tetB (57%) and 8 tetA (35%) were detected. All 24 ciprofloxacin resistant strains tested positive for gyrA, but none for qnrA. Sequence analysis of the quinolone resistance-determining regions (QRDR) of gyrA of two ciprofloxacin resistant strains showed two point mutations (S83L, D87N). Computational docking studies revealed a decreased interaction of the drug with the target due to this mutation. In conclusion, high incidence of plasmids and antimicrobial resistance in E. coli carrying virulence genes and resistance markers for antimicrobial drugs in diarrheagenic patients of Bangladesh is alarming as lateral transfer of these virulence and resistance genes in other infectious agents can make them irresistible, and infection control more challenging in the days to come.

Qinghua Fang inhibits high-fat diet-induced non-alcoholic fatty liver disease by modulating gut microbiota.

Disturbance of the gut microbiota may play a critical role in the progression of nonalcoholic fatty liver disease (NAFLD) induced by high-fat diet (HFD). Changes in gut microbiota were analyzed in a rat HFD-induced NAFLD model following treatment with Qinghua Fang (QHF), a Chinese herbal formula currently used in the clinical practice of traditional Chinese medicine. Sixty Wistar rats were randomly divided into 6 groups (n=10): blank group [normal chow (NC) group], model group (HFD group), control group (BG group), Qinghua Fang high-dose group [QHF(H) group], QHF mid-dose group [QHF(M) group], QHF low-dose group [QHF(L) group]. The high, medium and low doses of QHF were used to intervene in the H, M, and L groups; the BG group was given berberine; the NC and HFD groups were given distilled water for 10 weeks. H&E staining, determination of serum liver function and blood lipid levels, and changes in the structure of rat intestinal flora through 16S rDNA denaturing gradient gel electrophoresis sequencing technology and ERIC-PCR fingerprinting were performed. The liver function and blood lipid levels of the rats in the HFD group were higher than those in the NC group; the alanine aminotransferase levels in the QHF-H group, QFH-M group and QHF-L group were lower than in the HFD group (P<0.05); the liver pathology of the QHF-M group and QHF-H group showed a small amount of fatty cell infiltration, but was significantly less than the hepatocyte inflammation and necrosis in the HFD group. The ERIC-PCR fingerprint and diversity analysis found that the composition of the intestinal flora of rats in the QHF-H group was significantly different from that of the NC and HFD groups. The flora of the QHF and NC groups was more diverse and richer than in the HFD group (P<0.05). QHF alleviated the liver dysfunction and increased blood lipid levels of NAFLD rats induced by HFD. It also effectively reduced the degree of liver steatosis and adjusted the number and structure of intestinal flora. Treatment with QHF had a significant effect on NAFLD.

Emergence of NDM-5 Producing Carbapenem-Resistant Klebsiella aerogenes in a Pediatric Hospital in Shanghai, China.

Background: Carbapenem-resistant Klebsiella aerogenes (CRKA) has posed a serious threat for clinical anti-infective therapy. However, the molecular characteristics of CRKA in Shanghai are rarely reported.Objective: This study aimed to investigate the resistance profiles, dissemination mechanism, and molecular characteristics of CRKA strains isolated from children in a pediatric hospital, Shanghai.Method: Fifty CRKA isolates were collected in 2019. Antimicrobial susceptibility of the strains was determined by broth microdilution method. The β-lactamases and outer membrane porin genes were characterized by polymerase chain reaction (PCR). Conjugation experiments were performed to determine the transferability of the plasmids. The plasmids were typed based on their incompatibility group using the PCR-based replicon typing method. Multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) were performed for the genetic relationship.Results: All CRKA strains showed high level of resistance to cephalosporins and carbapenems, but still susceptible to aminoglycosides, colistin, and tigecycline. Forty five of fifty isolates carried blaNDM−5 genes (45/50, 90%), alongside with other β-Lactamase genes including blaCTX−M−1, blaTEM−1, and blaSHV−11 being detected. Loss of ompK35 and ompK36 genes were observed in 14% (7/50) and 28% (14/50), respectively, with 5 isolates lacking both ompK35 and ompK36. MLST analysis demonstrated that the majority of isolates belonged to ST4 (47/50, 94%) and ERIC-PCR fingerprinting was performed to identify NDM-5-producing isolates with approximately or more than 80% similarity levels. Plasmids carrying blaNDM−5 were successfully transferred to the E. coli recipient and plasmid typing showed that IncX3 were the prevalent among CRKA isolates.Conclusions: Our finding revealed the emergence of NDM-5 producing CRKA belonging to ST4 among children in Shanghai. Further attention should be paid to control the horizontal spread of the Class B carbapenemases like NDM in children.

Nodulation of Retama species by members of the genus Microvirga in Morocco

The genetic diversity of bacterial populations’ nodulating Retama sphaerocarpa grown in the soils of Maamora cork forest (Morocco) was examined. ERIC-PCR fingerprinting of 30 strains distributed them in 2 groups, of which a representative strain from each group was studied by multilocus sequence analysis of the 16S rRNA, atpD, and gyrB genes. The two representative strains RSM25 and RSM32, grouped with “Microvirga tunisiensis”. This is the first description of Retama nodule bacteria as members of the genus Microvirga. A nodC-based phylogeny confirmed that the two representative strains RSM25 and RSM32 are affiliated with symbiovar mediterranense. The 2 strains were capable of nodulating not only R. sphaerocarpa but also R. monosperma, R. dasycarpa and L. luteus, and unable to nodulate Phaseolus vulgaris, Vachellia gummifera, Cicer arietinum, Vigna unguiculata and Glycine max. The inoculation of R. sphaerocarpa with RSM25 or RSM32 produced a 1.22-, and 1.36-fold increase in the dry weight of the plants compared to those grown in the presence of 0.05% KNO3. The 2 strains used monosaccharides and disaccharides as the sole C source, but fructose, glucose, galactose, arabinose and starch were not used. They were unable to grow on glycine as a N source. Phosphate solubilization and siderophore production was not detected, but IAA or IAA-related compounds were produced. The alkaline pH of the sampling site soil in the Maamora forest where Retama grows could explain why Microvirga was the dominant species in the root nodules of the plants.

Putative novel Bradyrhizobium and Phyllobacterium species isolated from root nodules of Chamaecytisus ruthenicus

In this study, the diversity and the phylogenetic relationships of bacteria isolated from root nodules of Chamaecytisus ruthenicus growing in Poland were investigated using ERIC-PCR fingerprinting and by multilocus sequence analysis (MLSA). Two major clusters comprising 13 and 3 isolates were detected which 16S rRNA gene sequencing identified as Bradyrhizobium and Phyllobacterium. The results of phylogenetic analysis of individual and concatenated atpD, gyrB and recA gene sequences showed that the studied strains may represent novel species in the genera Bradyrhizobium and Phyllobacterium. In the phylogenetic tree based on the atpD-gyrB-recA concatemers, Bradyrhizobium isolates were split into two groups closely related to Bradyrhizobium algeriense STM89T and Bradyrhizobium valentinum LmjM3T. The genus Phyllobacterium isolates formed a separate cluster close to Phyllobacterium ifriqiyense LMG27887T in the atpD-gyrB-recA phylogram. Analysis of symbiotic gene sequences (nodC, nodZ, nifD, and nifH) showed that the Bradyrhizobium isolates were most closely related to Bradyrhizobium algeriense STM89T, Bradyrhizobium valentinum LmjM3T and Bradyrhizobium retamae Ro19T belonging to symbiovar retamae. This is the first report on the occurrence of members of symbiovar retamae from outside the Mediterranean region. No symbiosis related genes were amplified from Phyllobacterium strains, which were also unable to induce nodules on C. ruthenicus roots. Based on these findings Phyllobacterium isolates can be regarded as endophytic bacteria inhabitating root nodules of C. ruthenicus.

Astragalus algarbiensis is nodulated by the genistearum symbiovar of Bradyrhizobium spp. in Morocco

Astragalus algarbiensis is a wild herbaceous legume growing in Maamora, the most important cork oak forest in northern Africa. It is a plant of great importance as fodder in silvopastoral systems, and in the restoration of poor and degraded soils. The purpose of this study was to describe the biodiversity of rhizobia nodulating this plant and determine their identity. Out of 80 bacterial isolates, 56 strains isolated from root nodules of A. algarbiensis were characterized. ERIC-PCR fingerprinting grouped the strains in two main clusters containing 29 and 27 isolates, respectively, and the amplified ribosomal DNA restriction analysis (ARDRA) generated two different ribotypes. Based on both the ERIC-PCR and ARDRA results, representative strains As21 and As36 were selected for further genetic studies. The nearly complete 16S rRNA gene sequences of As21 and As36 showed that they were closely related to Bradyrhizobium cytisi CTAW11T with similarity values of 99.84% and 99.77%, respectively. Concatenation of atpD, recA, gyrB and dnaK housekeeping gene sequences indicated that strains As21 and As36 had a 95.22% similarity but they showed values of 95.80% and 94.97% with B. cytisi CTAW11T, respectively. The sequencing of the symbiotic nodC gene of the two strains revealed 97.20% and 97.76% identities, respectively, with that of B. cytisi CTAW11T isolated from Cytisus villosus growing in the Moroccan Rif Mountains. Furthermore, the phylogenic analysis showed that the strains isolated from A. algarbiensis clustered with B. cytisi and B. rifense within the bradyrhizobia genistearum symbiovar and may constitute two novel genospecies.

PREVALENCE OF ANTIBIOTIC RESISTANT V. parahaemolyticus AND V. cholerae IN FISH AND HUMANS WITH SPECIAL REFERENCE TO VIRULOTYPING AND GENOTYPING OF V. parahaemolyticus

This study aimed to investigate prevalence, virulence determinants, antibiogram and genotyping of Vibrio isolates from retail shrimp and tilapia fish as well as stool samples from gastroenteritis patients in Sharkia Governorate, Egypt. Vibrio spp were molecularly confirmed in 25.5% and 3% of fish and human stool samples, respectively. V. parahaemolyticus was isolated from 8.9%, 5% and 3% of shrimp, tilapia and stool samples, respectively. However, 0.7% of shrimp and 1.7% of tilapia were found to harbor V. cholera. trh and tdh virulence related genes were assessed in 34 V. parahaemolyticus isolates (25 from shrimp, 6 from tilapia and 3 from human stool). The tdh gene alone was recorded in 4 (16%) isolates from shrimp and 2 (66.7%) isolates from human stool. However, trh gene was detected alone in one (4%) isolate from shrimp. Moreover, both genes were detected simultaneously in one shrimp (4%) and one human stool (33.3%). Tilapia fish isolates were negative for both virulence genes. The resistance of the examined isolates were 100% (each of nalidixic acid and erythromycin), 81.6% (sulphamethoxazol), 73.7% (chloramphenicol), However, susceptibilities to gentamicin (81.6%), ciprofloxacin (73.7%) and 71.1% for each ampicillin/sulbactam and amikacin were observed. Multiple drug resistance was recorded in V. parahaemolyticus and V. cholerae isolates. Out of 38 isolates, 6 (15.8%) were resistant to all 14 antibiotics with MAR index of 1. Twenty of the isolates (52.6%) were resistant to 5-13 drugs with MAR index higher than 0.286. ERIC-PCR fingerprinting revealed five distinct profiles namely E1-E5 and the discriminatory index of the reaction was 0.5107, indicating low discrimination of the technique. In conclusion, this study revealed the contamination of tilapia and shrimp in fish markets with potentially virulent V. parahaemolyticus strains in the study area. Moreover, the presence of human and fish isolates in the cluster indicated the potential of the environmental isolates to cause human infection.

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students

BackgroundIn recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited.MethodsTwo hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality.ResultsAmong the 275 students, 55 (20%) carried a total of 56 E. coli (n = 35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study.ConclusionThis study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption.

Highly Diversified Pandoraea pulmonicola Population during Chronic Colonization in Cystic Fibrosis.

Several environmental bacteria are considered as opportunistic pathogens in cystic fibrosis (CF) and are able to persistently colonize the CF respiratory tract (CFRT). Beside Pseudomonas aeruginosa and Burkholderia cepacia complex, Pandoraea spp. are defined as pathogenic. During chronic colonization, adaptive evolution and diversified population have been demonstrated, notably for P. aeruginosa. However, the persistence of Pandoraea in the CFRT remains largely unexplored. We studied genomic and phenotypic traits of Pandoraea pulmonicola isolates successively recovered from the airways of a single CF patient and relate the results to qualitative and quantitative evolution of other cultivable pathogens and to patient clinical status. A total of 31 isolates recovered from 18 sputum samples over a 7-year period in a single CF patient were studied. Genome dynamics was assessed by pulsed-field gel electrophoresis, ERIC-PCR fingerprinting and 16S rRNA gene PCR-temporal temperature gel electrophoresis. Phenotypic features included antimicrobial susceptibility, motility, biofilm production, and virulence in Caenorhabditis elegans model. Variability was observed for all the characteristics studied leading to highly diversified patterns (24 patterns) for the 31 clonally related isolates. Some of these modifications, mainly genomic events were concomitantly observed with CFRT microbiota composition shifts and with severe exacerbations. The diversity of P. pulmonicola population studied, observed for isolates recovered from successive samples but also within a sample suggested that existence of a diversified population may represent a patho-adaptive strategy for host persistence in the heterogeneous and fluctuating CFRT environment.

Genomic fingerprints of Escherichia coli strains isolated from surface water in Alborz province, Iran

BackgroundConsistent use of suitable diagnostic methods is essential to evaluate the genomic diversity of E. coli strains. Advance of efficient methods to discriminate the causes of E. coli in aquatic environments is important. This study aimed to describe the strain diversity of an E. coli population retrieved from surface water.MethodsOne hundred water samples were drawn within a period of 1 year, from May 2012 to May 2013, and E. coli bacteria have been isolated from water samples. The genomic diversity analysis of 100 isolates of E. coli (one isolate per sample) has been carried out with the use of the ERIC-PCR fingerprinting method.ResultsOverall, our data indicated that complex fingerprint patterns have been obtained for totally of the isolates. Highest number of strains were in E4 (20 strains with more than 20% similarity) and lowest number of strains were in E3 (5 strains) group. In addition, there was no similarity in E1 (9 strains), E8 (10 strains) and E9 (7 strains) clusters.ConclusionTherefore, the occurrence of potential pathogenic E. coli and diversity of E. coli strains in surface water in Alborz province, Iran could pose a possible risk to animal health and human if not disinfected well.

Sulfitobacter pontiacus subsp. fungiae subsp. nov., Isolated from Coral Fungia seychellensis from Andaman Sea, and Description of Sulfitobacter pontiacus subsp. pontiacus subsp. nov.

Two closely related aerobic, Gram reaction-negative rod-shaped bacteria (S7-75T and S7-80) were isolated from mucus of coral Fungia seychellensis from Andaman Sea, India. Heterotrophic growth on marine agar was observed at 4-35 °C and pH 6.5-10.5; optimum growth occurred at 25-30 °C and pH 7-8. 16S rRNA sequence analysis confirmed the strains belonged to the genus Sulfitobacter and the two isolates shared more than 99.28% pairwise sequence similarity. DNA-DNA similarity between two isolates S7-75T and S7-80 was above 96%. Strain S7-75T showed maximum 16S rRNA similarity of 99.64% with Sulfitobacter pontiacus LMG 19752T. However, DNA-DNA relatedness between strain S7-75T and S. pontiacus LMG 19752T confirmed the placement of strain S7-75T as subspecies under the species S. pontiacus. Further, pulsed-field gel electrophoresis (PFGE), REP-PCR, ERIC-PCR fingerprint patterns and lipid profiles also differentiated strain S7-75T from the reference strain of S. pontiacus LMG 19752T. The DNA G+C content was 59.8mol%. Q10 was the major respiratory quinone. Based on polyphasic analysis, the isolate S7-75T represents a subspecies of S. pontiacus for which the name S. pontiacus subsp. fungiae subsp. nov. is proposed with S7-75T (=JCM 31094T = LMG 29158T) as type strain.