ERIC-PCR Profiles Research Articles

Molecular typing of clinical and non-clinical <i>Escherichia coli</i> from Rivers State, Nigeria using amplification-based RAPD and ERIC-PCR typing methods

Due to the diverse nature of Escherichia coli, typing of these organisms is essential. This can provide key epidemiological information not just on the strains in circulation and their relationship, but also on the development and spread of drug resistant clones. Affordable PCR-based typing techniques have resulted in an increase in typing studies particularly in resource limited settings. But information from Nigeria on strain typing is limited. This study was therefore aimed at typing clinical and non-clinical strains of E. coli from Nigeria using the PCR based Random Amplification of Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) typing methods. 
 E. coli was isolated from clinical and non-clinical sources using Eosin Methylene Blue agar. Characteristic green metallic sheen colonies associated with E. coli were purified, identities determined using standard biochemical tests and confirmed using molecular methods. Susceptibility testing was carried out using the modified Kirby-Bauer disc diffusion test and RAPD and ERIC-PCR typing carried out on 48 confirmed E. coli isolates as previously described. ERIC-PCR typing resulted in the generation of 38 unique patterns with a 0.94 diversity. Nine of these were singletons comprised of only a single isolate, while the remaining 29 patterns were grouped to 10 clusters. RAPD gave similar results of 37 patterns, 9 singletons, 0.94 diversity and 13 clusters. For ERIC-PCR, all isolates with identical patterns were from the same source. RAPD, however, had identical patterns present in isolates from different sources. 
 Using the ERIC-PCR typing method, this study was able to identify non-clinical isolates as distinct from the clinical E. coli isolates as evidenced by 100% identical ERIC-PCR profiles of isolates from the same source and a lack of 100% relatedness in isolates from different sources.

Evaluation of phenotypical and genotypical methods for the identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility.

Evaluate methods for identification and typing of Stenotrophomonas maltophilia isolated from a pharmaceutical facility. From 270 S. maltophilia strains identified by VITEK®2, 40 were selected and submitted to MALDI TOF-MS, 16S and 23S rRNA gene analysis, ERIC-PCR, and antimicrobial susceptibility profile. 16S rRNA sequencing was able to identify 39 (97.5%) strains as Stenotrophomonas spp. and one (2.5%) as Luteimonas huabeiensis. MALDI TOF-MS identified 37 (92.5%) strains as S. maltophilia, and three (7.5%) were not identified. PCR targeting 23S rRNA yielded a positive result for 39 (97.5%) strains. However, after sequencing, two strains were identified as Stenotrophomonas rhizophila, showing false-positive results. The confirmed S. maltophilia strains (n=37) showed 35 distinct ERIC-PCR profiles and exhibited sensitivity to minocycline and levofloxacin, and six (16.3%) showed intermediate resistance to sulfamethoxazole-trimethoprim. MALDI-TOF MS was a satisfactory methodology for the identification of S. maltophilia, but expansion of the database is necessary for the identification of other species. 16S rDNA sequencing showed low resolution for Stenotrophomonas species differentiation. PCR targeting 23S rRNA could not differentiate S. maltophilia from S. rhizophila. ERIC-PCR was shown to be a useful tool for the microbial source tracking of S. maltophilia.

Molecular detection and characterization of Shigella spp. harboring extended-spectrum β-lactamase genes in children with diarrhea in northwest Iran.

Shigellosis is one of the acute bowel infections and remains a serious public health problem in resource-poor countries. The present study aimed to survey the distribution of extended-spectrum β-lactamase (ESBL)-producing Shigella strains isolated from patients with diarrhea in northwest Iran. In the present cross-sectional study, from January 2019 to December 2020, 1280 fecal samples were collected from children with diarrhea in Ardabil, Iran. Multiplex PCR assay was applied for the presence of ipaH, invC, wbgZ, rfpB, and rfc genes to detect Shigella spp., Shigella sonnei, Shigella dysenteriae, Shigella flexneri, and Shigella boydii, respectively. Phenotypic detection of ESBL-producing isolates was carried out using the Double Disc Test (DDT). The frequency of main ESBL encoding genes including blaCTX-M, blaSHV, and blaTEM was detected using multiplex PCR. The genetic similarity of S. sonnei isolates was determined using ERIC PCR. A total of 49 Shigella isolates (3.8%; 49/1280) including 42 (85.7%) S. sonnei, 5 (10.2%) S. flexneri, and 2 (4%) S. dysenteriae were identified. S. boydii was not detected in any fecal samples. ESBLs were produced by 10.2% of Shigella spp. including 3 S. sonnei, 1 S. flexneri, and 1 S. dysenteriae. The ESBL encoding genes include blaCTX-M and blaTEM found in 65.3% and 61.2% of isolates, respectively. blaSHV gene was not detected in any isolates. The ERIC-PCR profiles allowed the differentiation of 42 S. sonnei strains into 6 clusters. Our study revealed a high frequency of ESBL-encoding genes among Shigella spp. in northwest Iran. The high prevalence of S. sonnei harboring ESBL genes, in the present work, is the main challenge for dysentery treatment, and this concern justifies the need for effective and regular monitoring of antibiotic usage among patients.

Co-existence of two Yersinia ruckeri biotypes and serotype O1a retrieved from rainbow trout (Oncorhynchus mykiss) farmed in Puno, Peru.

Yersinia ruckeri causes important economic losses for rainbow trout (Oncorhynchus mykiss) farms worldwide. This bacterial disease is likely the most common among trout in Peru; however, no commercial vaccine is available nationally, which is, in part, due to a lack of information on the bacterium. The aim of the current study was to characterize 29 Y. ruckeri isolates sampled from seven cage-reared farms in the Puno Region, the focal point for aquaculture activities in Peru. For this, samples were taken from fish with clinical signs (i.e. haemorrhages, uni- or bilateral exophthalmia, hyphaemia and/or melanosis). Notable among our findings was the existence of both Y. ruckeri biotype 1 (9 isolates) and biotype 2 (20 isolates; negative for sorbitol and Tween 80). The isolates further differed in API profiles 5307100 (21 isolates), 1307100 (4 isolates), 1305100 (2 isolates), 1307120 (1 isolate) and 5305100 (1 isolate), with the main differences being in the tests for lysine decarboxylase, gelatine hydrolysis and D-saccharose fermentation. Despite these differences, all isolates shared identical ERIC-PCR and REP-PCR profiles and belonged to the O1a serotype. Fingerprints were identical to the reference strain CECT 955 (serotype O1a). The information obtained will be used for epidemiological purposes by health authorities and for the development of a vaccine against Y. ruckeri, a prominent request made by fish farmers in Peru.

Presence of Clostridioides difficile in cattle feces, carcasses, and slaughterhouses: Molecular characterization and antibacterial susceptibility of the recovered isolates

The aims of this study were to isolate and identify Clostridioides difficile from cattle feces and carcasses, and slaughterhouse samples, and to determine the molecular characteristics and antibacterial susceptibility of the recovered isolates. A total of 220 samples, including 100 cattle fecal samples, 100 cattle carcass surface samples, and 20 slaughterhouse samples were used as the study material. In total, 12 (5.45%) samples, including 11 (11%) cattle fecal samples and 1 (5%) slaughterhouse sample, were found to be positive for C. difficile. On the other hand, all of the carcass samples were negative for C. difficile. A total of 11 (91.66%) isolates, including 10 fecal isolates and 1 slaughterhouse wastewater isolate, were found to be positive for the presence of the toxin genes tcdA and tcdB, whilst 1 fecal isolate was found to be negative for both genes. In addition, 3 different ERIC-PCR profiles were identified in the 11 fecal isolates. The ERIC-PCR profile of the slaughterhouse wastewater isolate was found to be similar to one of the ERIC-PCR profiles obtained from the fecal isolates. All of the isolates were resistant to ciprofloxacin and levofloxacin. Considering that the agent is a spore-forming bacterium shed in feces, the detection of C. difficile isolates of different genotypes, some carrying toxin genes, suggests that feces and slaughterhouse wastewater carrying this bacterium may pose a risk for the contamination of carcasses. The current study revealed that hygiene conditions should be performed to the maximum extent in slaughterhouses.

High prevalence of plasmid-mediated quinolone resistance (PMQR) among E. coli from aquatic environments in Bangladesh.

Fluro(quinolones) is an important class of antibiotic used widely in both human and veterinary medicine. Resistance to fluro(quinolones) can be acquired by either chromosomal point mutations or plasmid-mediated quinolone resistance (PMQR). There is a lack of studies on the prevalence of PMQR in organisms from environmental sources in Bangladesh. In this study, we investigated the occurrence of PMQR genes in E. coli from various water sources and analysed associations between multi-drug resistance (MDR) and resistance to extended spectrum β-lactam antibiotics. We analysed 300 E. coli isolates from wastewaters of urban live-bird markets (n = 74) and rural households (n = 80), rural ponds (n = 71) and river water samples (n = 75) during 2017-2018. We isolated E. coli by filtering 100 ml of water samples through a 0.2μm cellulose membrane and incubating on mTEC agar media followed by identification of isolated colonies using biochemical tests. We selected one isolate per sample for detection of PMQR genes by multiplex PCR and tested for antibiotic susceptibility by disc diffusion. Clonal relatedness of PMQR-positive isolates was evaluated by enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). About 66% (n = 199) of E. coli isolates harbored PMQR-genes, predominantly qnrS (82%, n = 164) followed by aac(6')-lb-cr (9%, n = 17), oqxAB (7%, n = 13), qnrB (6%, n = 11) and qepA (4%, n = 8). Around 68% (n = 135) of PMQR-positive isolates were MDR and 92% (n = 183) were extended spectrum β-lactamase (ESBL)-producing of which the proportion of positive samples was 87% (n = 159) for blaCTX-M-1' 34% (n = 62) for blaTEM, 9% (n = 16) for blaOXA-1, blaOXA-47 and blaCMY-2, and 2% (n = 4) for blaSHV. Further, 16% (n = 32) of PMQR-positive isolates were resistant to carbapenems of which 20 isolates carried blaNDM-1. Class 1 integron (int1) was found in 36% (n = 72) of PMQR-positive E. coli isolates. PMQR genes were significantly associated with ESBL phenotypes (p≤0.001). The presence of several PMQR genes were positively associated with ESBL and carbapenemase encoding genes such as qnrS with blaCTXM-1 (p<0.001), qnrB with blaTEM (p<0.001) and blaOXA-1 (p = 0.005), oqxAB and aac(6')-lb-cr with blaSHV and blaOXA-1 (p<0.001), qnrB with blaNDM-1 (p<0.001), aac(6')-lb-cr with blaOXA-47 (p<0.001) and blaNDM-1 (p = 0.002). Further, int1 was found to correlate with qnrB (p<0.001) and qepA (p = 0.011). ERIC-PCR profiles allowed identification of 84 of 199 isolates with 85% matching profiles which were further grouped into 33 clusters. Only 5 clusters had isolates (n = 11) with identical ERIC-PCR profiles suggesting that PMQR-positive E. coli isolates are genetically heterogeneous. Overall, PMQR-positive MDR E. coli were widely distributed in aquatic environments of Bangladesh indicating poor wastewater treatment and highlighting the risk of transmission to humans and animals.

Studies of Phylogeny, Symbiotic Functioning and Ecological Traits of Indigenous Microsymbionts Nodulating Bambara Groundnut (Vigna subterranea L. Verdc)in Eswatini.

Rhizobial microsymbionts of grain legumes are ubiquitous in soils and exhibit a wide range of diversity with respect to colony morphology, genetic variability, biochemical characteristics, and phylogenetic relationships. This study assessed the phylogenetic positions of rhizobial microsymbionts of Bambara groundnut from Eswatini exhibiting variations in morpho-physiology, adaptive characteristics, and N2-fixing efficiency. Theisolates' ERIC-PCR profiles revealed the presence of high genetic variation among them. These test isolates also exhibited differences in pH tolerance and IAA production. Multilocus sequence analysis based on the 16S rRNA, atpD, glnII, gyrB, and recA gene sequences of representative test isolates closely aligned themto the type strains ofBradyrhizobium arachidis, B. manausense, B. guangdongense, B. elkanii, and B. pachyrhizi. However, some isolates showed a high divergence from the known reference type strains, indicating that they may represent species yet to be properly characterized and described. Functional characterization in the glasshouse revealed that most of the isolates from the contrasting Agro-ecologies of Eswatini were efficient in N2 fixation, and therefore elicited greater stomatal conductance and photosynthetic rates in the homologous Bambara groundnut. Of the 75 isolates tested, 51% were more effective than the commercial Bradyrhizobium sp.strain CB756, with relative symbiotic effectiveness ranging from 138 to 308%. The findings of this study indicated that the analysis ofhousekeeping genes and functional traits of Bambara-nodulating microsymbionts can provide a clear view for understanding and predicting rhizobial community structure across environmental gradients.

Spreading of NDMI-Producing &lt;i&gt;Klebsiella&lt;/i&gt;&lt;i&gt; &lt;/i&gt;&lt;i&gt;Pneumoniae&lt;/i&gt;in Different Wards at Assiut University Hospital

Spreading of NDMI-producing klebsiella pneumoniaehas become a great trouble in many Egyptian hospitalized patients. Localizing the source of these isolates is an important step to prevent their spread in this country. This study included 33 NDMI-producing klebsiella pneumoniae that collected from different clinical specimens of patients admitted to different wards at Assiut University Hospitals in Egypt and confirmed to produce blaNDM1 gene by polymerase chain reaction. Isolates were typed by Enterobacterial Repetitive Intergenic Consensus (ERIC). Respiratory samples (51%) were the predominant samples and chestdepartment was the major ward (48.48%) that isolates recovered. ERIC gel profile showed that several identical isolates were found especially in chest ward and between different medical wards of this hospital suggested clonal transmission of NDMI-producing K. pneumoniae. Also, there are differences in the number and size of ERIC-PCR profiles indicated the genetic diversity among NDMI-producing K. pneumoniae isolates. The results of this study may help in tracing and controlling NDM-1-producing K. pneumoniae outbreak by applying effective infection control measures especially in the chest ward of this hospital.

High Frequency of 16S Ribosomal RNA Methyltransferases among Klebsiella pneumoniae Isolates: First Report of rmtA, rmtD, rmtE and rmtF Resistance Genes in Iran

Aims16S ribosomal RNA methyltransferases (RMTases) confer high-level resistance to aminoglycosides and are increasingly reported among Gram-negative bacilli, especially Klebsiella pneumoniae isolates. The objectives of the present study were to assess the resistance to aminoglycosides, the presence of RMTase genes, and the multilocus sequence typing (MLST) in urinary K. pneumoniae isolates.Materials & MethodsIn this study, 100 K. pneumoniae isolates were tested for susceptibility to amikacin and gentamicin by broth microdilution test according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Then the prevalence of RMTase genes was determined, and alleles and ST type of two selected isolates were identified by MLST. Finally, the isolates were genetically typed using Enterobacterial Repetitive Intergenic Consensus (ERIC) method.FindingsBroth microdilution assay showed that resistance to amikacin and gentamicin was 70 and 52%, respectively. In addition, 40% of the strains were resistant to both aminoglycosides. Also, rmtC (59.8%) gene was the most common type of RMTase genes investigated, followed by rmtA (51.2%), rmtD (47.6%), rmtF (43.9%), rmtE (41.5%), armA (41.5%), and rmtB (7.3%). K. pneumoniae isolates were assigned into two sequence types: ST51 and ST149. Using ERIC-PCR method, 3-7 different bands and 21 ERIC-PCR profiles were detected among the studied isolates.ConclusionThe high frequency of aminoglycoside resistance and the increased presence of 16S RMTases in K. pneumoniae strains are of great concern in Iran. Molecular typing showed high genetic diversity among the studied isolates. However, ST51 and ST149 were reported for the first time in Iran and could be considered as emerging strains.

Colistin heteroresistance in carbapenem-resistant Acinetobacter baumannii clinical isolates from a Thai university hospital.

Colistin is the last resort for the treatment of infections with carbapenem-resistant (CR) Gram-negative bacteria particularly Acinetobacter baumannii (CRAB). Currently, both colistin-resistant and -heteroresistant A. baumannii isolates have been reported globally. We therefore investigated the colistin heteroresistance rate in 75 non-duplicate colistin-susceptible CRAB clinical isolates from a Thai university collected in 2016. Minimum inhibitory concentrations (MICs) of colistin for all isolates were determined by broth microdilution method and carbapenemase genes were detected by PCR methods. All isolates were genotyped by ERIC-PCR method and screened for colistin heteroresistance by modified population analysis profile (PAP) method. The colistin MIC range for the 75 isolates was 0.5–2 µg/mL, with MIC50 and MIC90 of 1 and 2 µg/mL, respectively. Thirty-three isolates (44%) were considered colistin-heteroresistant with subpopulations growing at 3–8 μg/mL of colistin. After three daily passages of the subpopulations on antibiotic-free medium, their colistin MICs ranged from 4 to > 32 µg/mL, with MIC50 and MIC90 of 32 and > 32 µg/mL, respectively. Eight different ERIC-PCR profiles were obtained among the 33 isolates and all carried blaOXA-23-like. The high rate of colistin heteroresistance in the CRAB isolates highlights the possibility of treatment failure of CRAB infections by colistin due to the selection of colistin-resistant subpopulations.

β-lactam and fluoroquinolone resistance in Enterobacteriaceae from imported and locally-produced chicken in Mozambique.

Plasmid-mediated resistance to β-lactam and fluoroquinolone antibiotics was investigated in Enterobacteriaceae isolated from retailed frozen chickens from Brazil, South Africa and Mozambique. Carcass swabs and the liquid thaw of 33 chickens from each of the three countries constituted the total sample size of 198. Isolates were identified by biochemical tests, antibiotic susceptibility was ascertained by the disc diffusion assay and β-lactamases were detected using the double-disk synergy test. PCR was used to detect the presence of blaCTX-M, blaSHV, blaTEM, blaCMY, blaMOX, blaFOX, blaDHA, qnrB, qnrD, qnrS and qepA genes. A random selection of CTX-M genes was sequenced. The 198 samples yielded 27 (13.6%) putative extended-spectrum β-lactamase (ESBL)-positive isolates, 15 from carcass swabs and 12 from the liquid thaw from 22 chickens with 19, 5 and 3 isolates from South African, Mozambican and Brazilian chicken, respectively. Isolates exhibited the following resistance: ampicillin 100%, ceftriaxone 89%, trimethoprim-sulfamethoxazole 78%, cefotaxime 74%, ciprofloxacin 70%, ceftazidime 67%, cefoxitin 22% and gentamicin 8%. The predominant putative ESBL gene was blaSHV (85%), followed by blaCTX-M (62.9%) and blaTEM (44.4%) whilst blaMOX and blaDHA were the most common pAmpC genes at 33.3%. The predominant plasmid-mediated fluoroquinolone-resistance gene was qepA (22.2%). DNA sequencing identified blaCTX-M-55/-79/-101/-164. ERIC-PCR profiles did not show strong evidence of clonality. The Mozambican population is exposed to a reservoir of plasmid-mediated, and hence mobile β-lactam and quinolone resistance genes via imported, and to a lesser extent, locally produced poultry. This presents a food safety concern.

DOP06 Non-invasive identification of adherent-invasive E. coli in patients with Crohn’s disease

Abstract Background Medications limiting the adhesion of ‘adherent and invasive E. coli’ (AIEC) represent potential strategies to treat Crohn’s disease (CD). However, the ileal AIEC identification is a time-consuming procedure, and the number of AIEC strains which colonise ileal CD mucosa remains unknown. There is an unmet need for non-invasive biomarkers to identify patients colonised by AIEC. We aimed to evaluate non-invasive biomarker of ileal AIEC colonisation in patients with CD. Methods This prospective and multi-centre study included CD patients requiring ileocoloscopy. Saliva, serum, stools and ileal biopsies were collected. Abundance and global invasive ability of ileal or faecal E. coli were performed. Isolated E. coli were characterised as AIEC or non-AIEC on I407 epithelial cells and THP1 macrophages. The ERIC-PCR profiles of ileal E. coli were performed. Ileal E. coli/CEACAM6 interaction was analysed by a yeast aggregation test and T84 assays (CEACAM6 protein expression, adhesion inhibition test with D-mannose). Quantification of serum anti-E. coli and ileal or salivary CEACAM6 was realised by ELISA. Results Overall, 102 CD patients were enrolled in this study and 25.8% of them exhibited ileal AIEC colonisation (AIEC+). The abundance and global invasive ability of ileal mucosa-associated E. coli were higher in AIEC+ CD patients compared with CD patients without AIEC (AIEC−) (p = 0.0065 and p = 0.0007, respectively). There was no difference between faecal abundance and invasive ability of E. coli between AIEC+ and AIEC− patients. The ERIC-PCR profiles of ileal E. coli showed that CD AIEC+ were for 78% of them colonised by not more than 2 clonal AIEC strains. In addition, AIEC were able to interact with CEACAM6 by binding D-mannose residues and to induce CEACAM6 expression in T84 cells (p = 0.0009 and p = 0.0185, vs. non-AIEC; respectively). This was also supported by adhesion inhibition test. Serum anti-E. coli level was higher for CD AIEC+ (vs. CD AIEC-). Ileal CEACAM6 level were positively correlated with abundance of ileal associated E. coli in AIEC+ patients (r = 0.4000; p = 0.0362) and with salivary CEACAM6 level (r = 0.4690; p &amp;lt; 0.0001). The non-invasive biomarker ‘serum anti-E.coli/salivary CEACAM6’ index was higher for CD AIEC+ (p = 0.0174; vs. CD AIEC-). A cut-off value &amp;lt; 1.34 × 10−6 eliminated the presence of ileal AIEC with a high negative predictive value (90% CI95% [69%–95%]). Conclusion Our study reported that identification of faecal AIEC cannot replace identification of AIEC from ileal biopsies, most of AIEC infection are mono or bi-clonal (≤ 2 strains) and that non-invasive biomarker such as ‘serum anti-E.coli/salivary CEACAM6’ index could be helpful to screen CD patients for AIEC infection.

Incidence and risk of co-transmission of plasmid-mediated quinolone resistance and extended-spectrum β-lactamase genes in fluoroquinolone-resistant uropathogenic Escherichia coli: a first study from Kolkata, India.

Co-resistance to fluoroquinolones and β-lactams results in treatment complications for uropathogenic Escherichia coli (UPEC) infections. This study aimed to detect the coexistence and co-transmission of plasmid-mediated quinolone resistance (PMQR) and extended-spectrum β-lactamase (ESBL) genes in UPEC from Kolkata, India. Escherichia coli was detected biochemically from culture-positive urine samples. Antimicrobial resistance and ESBL production were confirmed by disk diffusion assay. Transfer of PMQR and ESBL genes was performed using azide-resistant E. coli J53 as recipient. PCR was conducted to identify PMQR and ESBL genes, plasmid incompatibility types, insertion sequences, integrons and ERIC-PCR patterns. PMQR determinants were detected in 50.0% (35/70) of ciprofloxacin-resistant isolates, with ESBL production in 42.9% (15/35) and a β-lactamase inhibitor-resistant phenotype in 51.4% (18/35). The highest co-occurrence (37.1%; 13/35) and co-transmission of aac(6')-Ib-cr with blaTEM, blaCTX-M and blaOXA was observed. Among the conjugal plasmids, replicon types FrepB/FrepB+F1B were predominant, with rare incidences of A/C, N, X, I1, FIIS, L/M and H1. Distribution of integrons and ISEcp1 and IS26, either alone or in combination, irrespective of PMQR and ESBL gene types was observed. Discrete ERIC-PCR profiles indicated that acquisition of PMQR and ESBLs and their dissemination may be attributed to horizontal gene transfer. This study demonstrates for the first time the risk of co-transmission of fluoroquinolone and β-lactam resistance amongst UPEC from Kolkata, posing a major public-health threat and limiting treatment options. Monitoring at the molecular level is necessary to design appropriate prescription policies to combat the alarming rise in drug resistance amongst these uropathogens.

Fluoroquinolone resistance in extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a Japanese tertiary hospital: silent shifting to CTX-M-15-producing K. pneumoniae.

Fluoroquinolone resistance (FQ-r) in extended-spectrum β-lactamase (ESBL) producers is an urgent health concern in countries where ESBL-producing K. pneumoniae (ESBL-Kpn) is prevalent. We investigated FQ-r in Japan where ESBL-Kpn is less prevalent. Clinical ESBL-Kpn isolates from 2011 to 2013 were collected in Nagasaki University Hospital. The ESBL genotypes included CTX-M-15, and the mechanisms of FQ-r through plasmid-mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining regions (QRDRs) were examined. Clonality was analysed by enterobacterial repetitive intergenic consensus (ERIC)-PCR and multi-locus sequence typing was performed on selected isolates.Results/Key findings. Thirty ESBL-Kpn isolates, including seven levofloxacin-resistant isolates, were obtained from different patients. An increase in CTX-M-15-producing strains was observed during the study period (0/11 in 2011, 3/8 in 2012, and 5/11 in 2013). PMQR was detected in 53.3 % of the isolates and aac-(6')-Ib-cr was the most common (36.7 %). ST15 was observed in 60.0 % of the isolates, and for the most predominant ERIC-PCR profiles, 62.5 % of the isolates possessed the CTX-M-15 genotype and 71.4 % were levofloxacin-resistant. Levofloxacin-resistance was significantly more common in CTX-M-15 isolates (62.5 %) compared to non-CTX-M-15 isolates (9.1 %). Three QRDR mutations and aac(6')-Ib-cr, but not qnrB and qnrS, were significantly enriched in the CTX-M-15 isolates (100.0 %) compared to the non-CTX-M-15 isolates (13.6 %). Cumulatively, these results indicate that the epidemic strain, the CTX-M-15-producing K. pneumoniae ST15, is covertly spreading even when ESBL producers are not prevalent. Monitoring these epidemic strains and ESBLs in general is important for quickly identifying health crises and minimizing future risks from FQ-r ESBL-Kpn.

Fingerprints of resistant Escherichia coli O157:H7 from vegetables and environmental samples.

Some routes of transmission of Escherichia coli O157:H7 to fresh produce include contaminated irrigation water and manure polluted soils. The aim of the present study was to determine the genetic relationships of E. coli O157:H7 isolated from some produce growing region in Nigeria using enterobacterial repetitive intergenic consensus (ERIC) DNA fingerprinting analysis. A total of 440 samples comprising leafy greens, irrigation water, manure and soil were obtained from vegetable producing regions in Kano and Plateau States, Nigeria. Genes coding for the quinolone resistance-determinant (gyrA) and plasmid (pCT) coding for multidrug resistance (MDR) were determined using polymerase chain reaction (PCR) in 16 isolates that showed MDR. Cluster analysis of the ERIC-PCR profiles based on band sizes revealed six main clusters from the sixteen isolates analysed. The largest cluster (cluster 3) grouped isolates from vegetables and manure at a similarity coefficient of 0.72. The present study provides data that support the potential transmission of resistant strains of E. coli O157:H7 from vegetables and environmental sources to humans with potential public health implications, especially in developing countries. © 2017 Society of Chemical Industry.

Clonal spread and accumulation of β-lactam resistance determinants in Enterobacter aerogenes and Enterobacter cloacae complex isolates from infection and colonization in patients at a public hospital in Recife, Pernambuco, Brazil.

Enterobacter aerogenes and Enterobacter cloacae complex are the two species of this genus most involved in healthcare-associated infections that are ESBL and carbapenemase producers. This study characterized, phenotypically and genotypically, 51 isolates of E. aerogenes and E. cloacae complex originating from infection or colonization in patients admitted to a public hospital in Recife, Pernambuco, Brazil, by antimicrobial susceptibility profile, analysis of β-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaKPC, blaVIM, blaIMP and blaSPM), PCR and DNA sequencing, plasmid profile and ERIC-PCR. In both species, the genes blaTEM, blaCTX-M and blaKPC were detected. The DNA sequencing confirmed the variants blaTEM-1, blaCTX-M-15 and blaKPC-2 in isolates. More than one gene conferring resistance in the isolates, including the detection of the three previously cited genes in strains isolated from infection sites, was observed. The detection of blaCTX-M was more frequent in isolates from infection sites than from colonization. The gene blaKPC predominated in E. cloacae complex isolates obtained from infections; however, in E. aerogenes isolates, it predominated in samples obtained from colonization. A clonal relationship among all of E. aerogenes isolates was detected by ERIC-PCR. The majority of E. cloacae complex isolates presented the same ERIC-PCR pattern. Despite the clonal relation presented by the isolates using ERIC-PCR, different plasmid and resistance profiles and several resistance genes were observed. The clonal dissemination and the accumulation of β-lactam resistance determinants presented by the isolates demonstrated the ability of E. aerogenes and E. cloacae complex, obtained from colonization and infection, to acquire and maintain different resistance genes.

Corynebacterium uropygiale sp. nov., isolated from the preen gland of Turkeys (Meleagris gallopavo)

A novel species of fastidious, lipophilic, club-shaped, Gram-positive bacteria was recovered from the preen glands of healthy Turkeys (Meleagris gallopavo) from two different locations. Phylogenetic analysis of the 16S rRNA gene showed highest similarity to Corynebacterium spheniscorum DSM 44757T (96.8%) with a 3.2kb stretch of rpoB sharing 82.4% sequence similarity to the same species. DNA fingerprinting by ERIC-PCR and polar lipid profiles clearly differentiated the Turkey isolates from the most closely related Corynebacteria, as did MALDI-TOF MS analysis. Chemotaxonomic tests revealed the presence of corynemycolic acids with C16:0, C18:0, C18:1ω9c and tuberculostearic acid as the major cellular fatty acids. The G+C content of the type strain was 60.7mol%. The species was susceptible to ampicillin, kanamycin A, streptomycin, amikacin, polymyxin B and vancomycin. From our results, it becomes evident that the isolated organisms represent a new species, for which the name Corynebacterium uropygiale sp. nov. is proposed. The type strain is Iso10T (=DSM 46817T=LMG 28616T).

Pectobacterium aroidearum and Pectobacterium carotovorum subsp. carotovorum as causal agents of potato soft rot in Lebanon

To identify bacteria causing severe soft rot on potato in Lebanon, pectinolytic bacteria were isolated from diseased samples collected in the Bekaa valley and the Akkar region, during 2009–2010. Six isolates that induced a hypersensitive reaction in tobacco plants and caused a marked soft rot when inoculated in potato tubers or stem, DAPP-PG 752 to DAPP-PG 757, were further characterized. Analysis of 16S–23S intergenic transcribed spacer-regions, digested with RsaI and CfoI, demonstrated that isolates DAPP-PG 753 to DAPP-PG 757 showed electrophoretic profiles similar to that of LMG 2408, a strain misclassified as Pectobacterium carotovorum subsp. carotovorum, and different from that of DAPP-PG 752. Phylogenetic analysis using partial malate dehydrogenase gene sequences, clustered DAPP-PG 752 in a clade that contained the type strain of P. carotovorum subsp. carotovorum, while DAPP-PG 753 to DAPP-PG 757, together with LMG 2408, fell in a clade including strains of the recently described species Pectobacterium aroidearum. Multilocus sequence analysis of concatenated partial sequences of the atpD, gyrB, infB and rpoB genes confirmed the results obtained with the mdh gene sequences and identified DAPP-PG 752 as P. carotovorum, and LMG 2408 and the five related Lebanese isolates as P. aroidearum. The Lebanese P. aroidearum isolates were phenotypically similar to known P. aroidearum strains, based on API 50 CHE data. They showed identical BOX- and ERIC-PCR profiles that resembled profiles of known P. aroidearum strains. This study represents the first official report of P. aroidearum as causal agent of soft rot on potato plants and the first occurrence of P. carotovorum subsp. carotovorum on potato plants in Lebanon.

Epidemiological and Inducible Resistance in Coagulase Negative Staphylococci.

Background and Objectives:Coagulase negative staphylococci (CNS) are potential pathogens with the increased use of implants in hospitals. Macrolide, lincosamide and streptogramin B (MLSB) are used in the treatment of staphylococcal infections. The aim of this study was to molecular detection of inducible clindamycin resistance and genetic pattern in CNS isolates and their transmission between hospitals.Materials and Methods:110 CNS strains, isolated from hospitalized patients in the intensive care unit and infectious wards of Besat and Toohid hospitals, Sanandaj. Methicillin resistance was done by agar screen test and the resistance inducible Clindamycin by the D-Test. Multiplex PCR was performed, using primers specific for erm (A, B, C, and TR) genes. Diversity of strains was determined by ERIC–PCR technique based on the similarities between DNA fingerprints by using Jaccards coefficient in the SAHN program of the NTSYS-pc software.Results:Of the 110 isolates, 64(58.2%) were methicillin -resistant CNS (MRCNS), 48(43.6%) were resistant to erythromycin (ERCNS). Out of 48 Erythromycin-resistant strains 5 (10.4%) were iMLSB phenotypes that 4 isolates showed genes erm by Multiplex PCR. The ERIC–PCR profiles allowed typing of the 110 isolates into 90 ERIC-types which were grouped into fourteen main clusters (C1–C14).Conclusion:The results of this study also showed that most of CNS isolated produced different genomic fingerprint patterns, therefore, source of infection is differen t.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR oneand these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8–100, 18–100, and 23-100 %, respectively.