Pulsed-field Gel Electrophoresis Analysis Research Articles

Genotypic and phenotypic analysis of Elizabethkingia meningoseptica in bullfrog Rana catesbeiana isolated in Taiwan.

Elizabethkingia meningoseptica is a hazardous bacterium for agriculture production and human health. The present study identified E. meningoseptica from the bullfrog, human and reference strain BCRC 10677 by API 20NE, 50S ribosome protein L27 sequencing and pulse field gel electrophoresis to differentiate isolates of E. meningoseptica from aquatic animals and humans. All isolates from bullfrogs and humans were identified as E. meningoseptica by DNA sequencing with 98.8%-100% sequence identity. E. meningoseptica displayed significant genetic diversity when analysed using pulsed-field gel electrophoresis (PFGE). There were six distinct pulsotypes, including one pulsotype found in bullfrog isolates and five pulsotypes found in human isolates. However, E. meningoseptica from bullfrog exhibited one genotype only by PFGE. Overall, molecular epidemiological analysis of PFGE results indicated that the frog E. meningoseptica outbreaks in Taiwan were produced by genetically identical clones. The bullfrog isolates were not genetically related to other E. meningoseptica from human and reference isolates. This research provided the first comparisons of biochemical characteristics and genetic differences of E. meningoseptica from human and bullfrog isolates.

Pulsed-Field Gel Electrophoresis Analysis of Bovine Associated Staphylococcus aureus: A Review.

For decades now, DNA fingerprinting by means of pulsed-field gel electrophoresis (PFGE) continues to be the most widely used to separate large DNA molecules and distinguish between different strains in alternating pulses. This is done by isolating intact chromosomal DNA and using restriction enzymes with specific restriction sites to generate less than 30 restriction fragments from 50 Kb to 10 Mbp. These results make clone-specific band profiles easy to compare. Specialized equipment is required for the optimization of DNA separation and resolution, among which a contour-clamped homogeneous electric field (CHEF) apparatus is the most commonly used. As a result, the PFGE analysis of a bacterial genome provides useful information in terms of epidemiological investigations of different bacterial pathogens. For Staphylococcus aureus subtyping, despite its limitations and the emergence of alternative methods, PFGE analysis has proven to be an adequate choice and the gold standard for determining genetic relatedness, especially in outbreak detection and short-term surveillance in the veterinary field.

Dissemination of ArmA- and OXA-23-co-producing Acinetobacter baumannii Global Clone 2 in Switzerland, 2020-2021.

Following the observation of an increased number of isolation of OXA-23- and ArmA-producing Acinetobacter baumannii at the national level, our aim was to evaluate whether some given clone(s) might actually be spreading and/or emerging in Switzerland. To evaluate this possibility, our study investigated and characterized all A. baumannii isolates harboring both the blaOXA-23 and armA genes that had been collected at the Swiss National Reference Center for Emerging Antibiotic Resistance (NARA) from 2020 to 2021. Most isolates were obtained from infections rather than colonization with the majority being obtained from respiratory specimens. Pulsed-field gel electrophoresis (PFGE) analysis of 56 isolates identified nine profiles. Then, whole-genome sequencing that was performed on a subset of 11 isolates including at least one representative isolate of each PFGE profile identified three STs; one each of ST25 and ST1902, and nine ST2 (a member of Global Clone 2 (GC-2). The blaOXA-23 gene was always found embedded within Tn2006 structures, as commonly described with GC-2 (ST2) isolates. Susceptibility testing showed that most of those isolates, despite being highly resistant to all carbapenems and all aminoglycosides, remained susceptible to colistin (94.6%), sulbactam-durlobactam (87.5%), and cefiderocol (83.9% or 91.1% according to EUCAST or CLSI breakpoints, respectively). Overall, this study identified that the A. baumannii co-producing OXA-23 and ArmA are increasing in incidence in Switzerland, largely due to the dissemination of the high-risk GC-2. This highlights the importance of themonitoring of such MDR A. baumannii strains, in order to contribute to reduce their potential further spread.

Antibiotic resistance and genetic diversity of Salmonella enterica serovar 1,4,[5],12:i:- isolated from animal-derived foods and humans in Shanghai, China

Salmonella enterica serovar 1,4,[5],12:i:- (S. 1,4,[5],12:i:-) is an emerging pathogen that poses a significant risk of foodborne outbreaks, particularly through the contamination of animal-derived foods. This study aimed to investigate the antibiotic susceptibility and genetic diversity of 32 S. 1,4,[5],12:i:- isolates from animal-derived foods and humans in Shanghai, China. The results revealed a high prevalence of resistance among these isolates, particularly against ampicillin (71.9%), tetracycline (65.6%), tobramycin (53.1%), trimethoprim-sulfamethoxazole (40.6%), and ciprofloxacin (40.6%). Furthermore, more than half of the isolates (56.3%) exhibited multidrug resistance. Pulsed-field gel electrophoresis analysis revealed ten distinct PFGE patterns. Several pork and clinical isolates grouped into the same pattern with 100% genetic similarity, indicating significant transmission of S. 1,4,[5],12:i:- through pork. Additionally, nine plasmid incompatibility groups (Inc) were identified, with IncQ1, IncHI2A, and IncHI2 being the most prevalent types. Interestingly, 15 isolates from foods and humans contained both IncHI2A-HI2 plasmids. These plasmids carried numerous resistance genes (e.g., blaCTX-M, blaTEM, blaOXA), suggesting their potential role in the transfer of resistance genes. The findings underscore the high prevalence of MDR S. 1,4,[5],12:i:- isolates in animal-derived foods and humans. Strengthening surveillance of animal-derived food isolates is crucial to prevent and control their spread in the food chain.

Tracking spatio-temporal distribution and transmission of antibiotic resistance in aquatic environments by using ESBL-producing Escherichia coli as an indicator

Wastewater treatment plants (WWTPs) play an important role in the production, and transmission of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) as a convergence for human, animal, and environmental wastewater. The aim of this study was to investigate the spatio-temporal variation and influencing factors of ARB in different functional areas of the urban WWTP and the connecting rivers for 1-year monitoring using extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec) as an indicator bacteria, and to study the transmission patterns of ARB in the aquatic environment. The results showed that ESBL-Ec isolates were identified from the WWTP (n = 219), including influent (n = 53), anaerobiotic tank (n = 40), aerobiotic tank (n = 36), activated sludge tank (n = 31), sludge thickner tank (n = 30), effluent (n = 16), and mudcake storage area (n = 13). The dehydration process can significantly remove the ESBL-Ec isolates; however, ESBL-Ec was still detected in samples collected from the effluent of the WWTP (37.0%). The detection rate of ESBL-Ec was significantly different across seasons (P < 0.05), and ambient temperature was negatively correlated with the detection rate of ESBL-Ec (P < 0.05). Furthermore, a high prevalence of ESBL-Ec isolates (29/187, 15.5%) was detected in samples collected from the river system. These findings emphasize that the high majority of ESBL-Ec in aquatic environments is alarming because it poses a significant threat to public health. Clonal transmission of ESBL-Ec isolates between the WWTP and rivers based on the spatio-temporal scale was observed by pulsed-field gel electrophoresis analysis, ST38 and ST69 ESBL-Ec clone were selected as prioritized isolates for antibiotic resistance monitoring in the aquatic environment. Further phylogenetic analysis showed human-associated (feces, blood) E. coli was the main source contributing to the presence of antibiotic resistance in aquatic environments. Longitudinal and targeted monitoring of ESBL-Ec in WWTPs and the development of effective wastewater disinfection strategies before effluent discharge from WWTPs are urgently required, to prevent and control the spread of antibiotic resistance in the environment.

Clonal diversity of Staphylococcus aureus isolates in clinical specimens from selected health facilities in Ethiopia

Staphylococcus aureus is among the top three causative agents of nosocomial infection in Ethiopia. The majority of studies in Ethiopia have focused on the epidemiology of S. aureus in hospital settings, with limited molecular genotyping results. Molecular characterization of S. aureus is essential for identification of strains, and contributes to the control and prevention of S. aureus infection. The aim of the current study was to determine the molecular epidemiology of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates recovered from clinical specimens in Ethiopia. A total of 161 MSSA and 9 MRSA isolates were characterized using pulsed-field gel electrophoresis (PFGE) and staphylococcal protein A (spa) typing. Based on the PFGE analysis, MSSA isolates were grouped into eight pulso-types groups (from A to I), while MRSA isolates clustered into three (A, B and C) pulso-types with more than 80% similarity. The spa typing analysis showed diversity of S. aureus with 56 distinct spa types. Spa type t355 was most prevalent (56/170, 32.9%), while eleven new spa types were detected including t20038, t20039, and t20042. The identified spa types were clustered into 15 spa-clonal complexes (spa-CCs) using BURP analysis; novel/unknown spa types were further subjected to MLST analysis. The majority of isolates belonged to spa-CC 152 (62/170, 36.4%), followed by spa-CC 121 (19/170, 11.2%), and spa-CC 005 (18 /170, 10.6%). Of the nine MRSA isolates, 2 (22.2%) were spa-CC 239 with staphylococcal cassette chromosome (SCC)mec III. These findings highlight the diversity of S. aureus strains in Ethiopia, as well as the presence of potentially epidemic strains circulating in the country necessitating further characterization of S. aureus for antimicrobial resistance detection and infection prevention purposes.

Staphylococcus aureus and CA-MRSA Carriage among Brazilian Indians Living in Peri-Urban Areas and Remote Communities.

The emergence of Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections among indigenous populations has been reported. Usually, indigenous communities live in extreme poverty and are at risk of acquiring infections. In Brazil, healthcare inequality is observed in this population. To date, there are no reports of CA-MRSA infections, and no active search for asymptomatic S. aureus carriage has been conducted among Brazilian Indians. The aim of this study was to investigate the prevalence of colonization with S. aureus and CA-MRSA among Brazilian Indians. We screened 400 Indians (from near urban areas and remote hamlets) for S. aureus and CA-MRSA colonization. The isolates were submitted to clonal profiling by pulsed-field gel electrophoresis (PFGE), and selected isolates were submitted to multilocus sequence typing (MLST). Among 931 specimens (nasal and oral) from different indigenous individuals in remote hamlets, S. aureus was cultured in 190 (47.6%). Furthermore, CA-MRSA was found in three isolates (0.7%), all SCCmec type IV. PFGE analysis identified 21 clusters among the S. aureus isolates, and MLST analysis showed a predominance of sequence type 5 among these isolates. Our study revealed a higher prevalence of S. aureus carriage among Shanenawa ethnicity individuals (41.1%). Therefore, ethnicity appears to be associated with the prevalence of S. aureus in these populations.

High prevalence of clonally related ST182 NDM-1-producing Enterobacter cloacae complex clinical isolates in Greece

NDM-type metallo-β-lactamase (MBL)-producing Enterobacterales remain uncommon in the European region, especially among species other than Klebsiella pneumoniae and Escherichia coli. The aim of this study was to describe epidemiological and molecular characteristics of a widespread NDM-1-producing Enterobacter cloacae complex outbreak in Greece. Over a 6-year period (March 2016-March 2022), a retrospective study was conducted in a tertiary care Greek hospital. Ninety single-patient carbapenem-non-susceptible E. cloacae complex clinical isolates were recovered consecutively. The isolates were subjected to further investigation, including antimicrobial susceptibility testing and combined disc tests for carbapenemase production, polymerase chain reaction and sequencing for resistance genes, molecular fingerprinting by pulsed-field gel electrophoresis (PFGE), plasmid profiling, replicon typing, conjugation experiments, genotyping by multi-locus sequence typing (MLST), whole-genome sequencing and phylogenetic analysis. Phenotypic and molecular testing confirmed the presence of blaNDM-1 in 47 (52.2%) of the E. cloacae complex isolates. MLST analysis clustered all but four of the NDM-1 producers into a single MLST sequence type (ST182), whereas single isolates belonged to different sequence types (ST190, ST269, ST443 and ST743). PFGE analysis revealed that ST182 isolates were clustered into a single clonal type, with three subtypes, which differed from the clonal types detected among the remaining carbapenem non-susceptible E. cloacae complex isolates identified during the study period. All ST182 blaNDM-1-carrying isolates also harboured the blaACT-16 AmpC gene, while the blaESBL, blaOXA-1 and blaTEM-1 genes were detected in most cases. In all clonal isolates, the blaNDM-1 gene was located on an IncA/C-type plasmid, and flanked upstream by an ISAba125 element and downstream by bleMBL. Conjugation experiments failed to produce carbapenem-resistant transconjugants, indicating a low dynamic for horizontal gene transfer. Application of enforced infection control measures led to the absence of new NDM-positive cases for periods of time during the survey. This study represents the largest clonal outbreak of NDM-producing E. cloacae complex in Europe.

Prevalence of Antimicrobial-resistant &lt;i&gt;Escherichia coli&lt;/i&gt; in Environmental Samples from Areas Inhabited by the Endangered &lt;i&gt;Hypotaenidia okinawae&lt;/i&gt;

Antimicrobial resistance (AMR) in pathogens has become a social problem across humans and wild animals. In particular, the environmental contamination caused by AMR should be monitored. In this study, we analyzed the prevalence of Escherichia coli and its AMR, as well as the distribution and genotype of E. coli in environmental soil and water samples from the habitat of the Hypotaenidia okinawae (Okinawa rail), to elucidate the potential of AMR pollution between the bird and its habitat. The habitat of Okinawa rail was divided into a livestock farm area (LA) near human settlements and a forest area (FA) as a non-human living environment. We found that the prevalence of E. coli in environmental samples collected from the LA was 38.2% (13/34), of which 84.6% (11/13) were antimicrobial resistant. In contrast, the prevalence of E. coli in environmental samples collected from the FA was 25.5% (12/47), of which 16.7% (2/12) were antimicrobial resistant. These results indicate that antimicrobial resistant E. coli was more prevalent in the LA than FA. Furthermore, pulsed-field gel electrophoresis analysis revealed a similar pattern of E. coli prevalence. This suggests that the occurrence of environmental pollution of AMR from livestock farms and that the Okinawa rail may become a carrier of antimicrobial-resistant E. coli in the Yambaru region of Okinawa.

Spectrum of ctxB genotypes, antibiogram profiles and virulence genes of Vibrio cholerae serogroups isolated from environmental water sources from Odisha, India

BackgroundThe present study reports on the comprehensive analysis of Vibrio cholerae O1 and non-O1/non-O139 serogroups isolated from environmental water sources during cholera outbreaks, epidemics and surveillance studies between years 2007 to 2019 from different districts of Odisha, India.MethodsA total of 85 stocked cultures of V. cholerae O1 and non-O1/non-O139 strains were analyzed for different ctxB genotypes, toxic genes, antibiogram profiles through PCR assays and pulsotyped by pulsed-field gel electrophoresis (PFGE).ResultsFrom all V. cholerae strains tested, 51 isolates were O1 Ogawa and the rest 34 strains were non-O1/non-O139. All the V. cholerae O1 strains were altered El Tor variants carrying ctxB1, ctxB3 and ctxB7 genotypes. However, only ctxB1 genotypes were present in V. cholerae non-O1/non-O139. Though non-O1/non-O139 strains were negative by O1 antisera, 20% strains were positive for rfbO1 gene by PCR assay. All the V. cholerae isolates possessed a variety of virulence genes including ace, ctxAB, toxR, zot, hlyA which were in higher percentage in the case of V. cholerae O1. The Vibrio cholerae O1 and non-O1-/non-O139 strains showed multiple antibiotic resistances in 2007 and 2012. The PCR detection of four resistance associated genes (strB, dfrA1, sulll, SXT) confirmed higher prevalence in V. cholerae non-O1/non-O139 strains. The PFGE analysis revealed 3 pulsotypes having 93% similarity among V. cholerae O1 strains.ConclusionThis study indicates the changing epidemiology, antibiogram patterns and continuous genetic variation in environmental V. cholerae strains of Odisha over the years. So continuous surveillance is necessary to understand the changing patterns of V. cholerae different serogroups isolated from stool and water samples from Odisha.

PFGE analysis of Listeria monocytogenes isolates from milk, bovine clinical and bovine environment origin from Nagpur

Listeria monocytogenes, a foodborne pathogen, causes listeriosis, that mainly affects immunocompromised persons. Epidemiological investigation and molecular subtyping can be helpful in linking the specific sources of infection. Thirty-eight isolates of Listeria monocytogenes recovered from the bovine environment, milk, and bovine clinical cases were characterized by serotyping and pulse field gel electrophoresis (PFGE) to correlate the genetic links between the isolates. Serotyping revealed 3 L. monocytogenes isolates to be of serovar group 4b, 4d and 4e, 34 isolates of serovar group 1/2b, 4b, 3b, 4d, 4e, and one isolate to be serovar group 1/2a, 1/2c,3a, and 3c. The PFGE analysis revealed 38 L. monocytogenes isolate from seven different clusters combined ApaI and AscI with 80% relative genetic similarity. In comparison majority of the strains showed location and serovar wise homology in PFGE pattern than source wise. It was also observed that at the place of collection, the organisms circulate in the environment. The present findings suggested that the dairy/bovine farm environment is a potential source of contamination of L. monocytogenes in milk and animals.

Molecular characterization of colistin resistance genes in a high-risk ST101/KPC-2 clone of Klebsiella pneumoniae in a University Hospital of Split, Croatia.

Klebsiella pneumoniae carbapenemase-producing K. pneumoniae (KPC-KP) has become a major concern worldwide due to multidrug resistance and the ability to spread locally and globally. Infections caused by KPC-KP are great challenge in the healthcare systems because these are associated with longer hospitalization and high mortality. The emergence of colistin resistance has significantly reduced already limited treatment options. This study describes the molecular background of colistin-resistant KPC-KP isolates in the largest hospital in southern Croatia. Thirty-four non-duplicate colistin-resistant KPC-KP isolates were collected during routine work from April 2019 to January 2020 and from February to May 2021. Antimicrobial susceptibility was determined using disk diffusion, broth microdilution, and the gradient strip method. Carbapenemase was detected with an immunochromatographic test. Identification of blaKPC and mcr genes or mutations in pmrA, pmrB, mgrB, phoP, and phoQ genes were performed by polymerase chain reaction (PCR) and positive products were sequenced. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were used for epidemiological analysis. All isolates were multidrug-resistant, with colistin minimum inhibitory concentrations (MICs) from 4 to >16 mg/L, and all harbored blaKPC-2 and had a single point mutation in the mgrB gene resulting in a premature stop codon, with the exception of one isolate with four point mutations corresponding to stop codons. All isolates were negative for mcr genes. PFGE analysis identified a single genetic cluster, and MLST revealed that all isolates belonged to sequence type 101 (ST101). These results show emergence of the high-risk ST101/KPC-2 clone of K. pneumoniae in Croatia as well as appearance of colistin resistance due to mutations in the mgrB gene. Molecular analysis of epidemiology and possible resistance mechanisms are important to develop further strategies to combat such threats.

Emergence and clonal spread of KPC-2-producing clinical Klebsiella aerogenes isolates in a hospital from northwest Argentina.

Introduction. Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and healthcare-associated infections.Gap Statement. K. aerogenes is associated with hospital-acquired infections with the ability to acquire mechanisms of resistance to reserve antimicrobials; its clinical behaviour has been poorly documented.Objective. We proposed to investigate an outbreak of carbapenem-resistant K. aerogenes in a hospital that persisted for 4 months.Methods. The primary aim was to evaluate the molecular characteristics and the clonal relationships among the isolates. We characterized isolates by polymerase chain reaction (PCR) and pulsed-field gel electrophoresis (PFGE). The information was integrated with clinical and epidemiological data.Results. Fourteen strains were disseminated in an intensive care unit and different wards at the hospital. The overall mortality was 42.8 %, and mortality attributed to infection was 21.4 %; strains showed high rates of resistance to most of the antimicrobials tested and carried bla KPC-2, bla SHV-2 and bla CTXM-15 genes. PFGE analysis indicated 2 PFGE groups; 12/14 isolates were associated with subgroup A and were likely to be primarily responsible for the first isolation and subsequent dissemination. The outbreak characteristics data showed prolonged hospitalization and previous use of antibiotics as potential risk factors.Conclusion. We consider that it is essential to perform phenotypic and genotypic identification of early genetic resistance mechanisms in K. aerogenes isolates, not only from infection sites but also from colonization, to prevent the spread of these multidrug-resistant (MDR) isolates.

The Epidemiological and Clinical Characteristics of the Largest Outbreak of Enterohemorrhagic Escherichia coli in Korea.

The largest outbreak of enterohemorrhagic Escherichia coli (EHEC) O157:H7 occurred at a preschool in South Korea from June 12 to 29, 2020. This study aimed to analyze the epidemiological and clinical characteristics of EHEC infection in this outbreak. Epidemiological investigation was performed on all 184 children and 19 workers at the preschool using a standard questionnaire to assess symptoms, food intake, attendance, and special activity history. Pulsed-field gel electrophoresis analysis of confirmed cases was performed to determine genetic relevance. During this outbreak, 103 children were affected, whereas only one infection was identified in adults. Of the 103 pediatric patients, 85 had symptoms (82.5%), including diarrhea, abdominal pain, bloody stool, fever, and vomiting. Thirty-two patients (31.1%) were hospitalized, 15 (14.6%) were diagnosed with hemolytic uremic syndrome, and 4 (3.9%) received dialysis treatment. Pulsed-field gel electrophoresis analysis identified 4 genotypes with high genetic relevance (92.3%). Epidemiological investigation revealed that this outbreak might have occurred from ingesting foods stored in a refrigerator with a constant temperature above 10°C, which was conducive to bacterial growth. Despite several measures after outbreak recognition, new infections continued to appear. Therefore, the preschool was forced to close on June 19 to prevent further person-to-person transmission. Our findings from the response to the largest outbreak will help prepare countermeasures against future EHEC outbreak.

Emergence of OptrA Gene Mediated Linezolid Resistance among Enterococcus Faecium: A Pilot Study from a Tertiary Care Hospital, India.

E. faeciumis the third most common cause of nosocomial infections. Linezolid (LNZ) is a reserve antibiotic recommended for infections caused by vancomycin resistant E. faecium (VREfm).The aim of the present study was to investigate the prevalence of optrAgene among linezolid resistant E. faecium (LREfm) and to study the molecular epidemiology using pulse field gel electrophoresis (PFGE).Clinically significant LREfm were identified and antimicrobial susceptibility was performed by disc diffusion. Minimum inhibitory concentration (MIC) of linezolid, vancomycin, daptomycin and quinupristin/dalfopristin was determined by E-test.PCR and PCR-RFPL were performed for the detection of optrA/cfrgene and G2576T mutation respectively. Molecular epidemiology was studied by PFGE. A total of 1081 clinically significant Enterococci species were isolated which included E. faecium 63.5% (n=687) and E. faecalis 36.5% (n=394). LREfm (30/687) were further studied. Multidrug resistance and vancomycin resistance was 100% and 80%, respectively. Linezolid MIC range was 8-256µg/ml and the most common mechanism of resistance was optrA gene (83.3%) followed by G2576T mutation (33.3%). PFGE analysis demonstrated 4 major clones. The optrAgene mediated linezolid resistance was high and PFGE suggests resistance was emerging in the different background strains irrespective of resistance mechanism. Studies are required to investigate factors driving the emergence of linezolid resistance. The review suggests that this is the first report of optrA-mediated resistance in E. faecium from India.

Genotypic characterization and clonal relatednessof metallo-β-lactamase-producingnon-fermentative gram negative bacteria in the first 5years of their circulation in Paraguay(2011-2015).

Pseudomonas aeruginosa and species of Acinetobacter calcoaceticus-baumanii complex are multiresistant intrahospital opportunistic pathogens, able to acquire carbapenemases and produce outbreaks with high morbidity and mortality. Pseudomonas putida has also emerged with similar characteristics. The aim of this research was to characterize the Metallo-β-lactamases (MBLs) detected by surveillance in Paraguay in the first 5years of their circulation in hospitals. The coexistence of KPC and OXA-type carbapenemases was also investigated. 70 MBL-producing strains from inpatients were detected from clinical samples and rectal swab from 11 hospitals. The strains were identified by manual, automated, and molecular methods. Antimicrobial susceptibility was studied by Kirby-Bauer and automated methods, while colistin susceptibility was determined by broth macrodilution. MBLs were investigated by synergy with EDTA against carbapenems and PCR, and their variants by sequencing. KPC and OXA-carbapenemases were investigated by PCR. Clonality was studied by pulsed-field gel electrophoresis (PFGE). The results demonstrated the circulation of blaVIM-2 (60%), blaNDM-1 (36%), and blaIMP-18 (4%). The MBL-producing species were P. putida (45.7%), P. aeruginosa (17.2%), A. baumannii (24.3%), A. pittii (5.7%), A. nosocomialis, (4.3%) A. haemolyticus (1.4%), and A. bereziniae (1.4%). PFGE analysis showed one dominant clone for A. baumannii, a predominant clone for half of the strains of P. aeruginosa, and a polyclonal spread for P. putida. In the first 5years of circulation in Paraguay, MBLs were disseminated as unique variants per genotype, appeared only in Pseudomonas spp. and Acinetobacter spp., probably through horizontal transmission between species and vertical by some successful clones.

Characterization of mammary pathogenic Escherichia coli reveals the diversity of Escherichia coli isolates associated with bovine clinical mastitis in Brazil

Mammary pathogenic Escherichia coli (MPEC) is one of the most common pathogens associated with clinical mastitis. We analyzed isolates obtained from milk samples of cows with clinical mastitis, collected from 10 farms in Brazil, to verify molecular and phenotypic characteristics. A total of 192 (4.5%) mammary pathogenic E. coli isolates were obtained from 4,275 milk samples analyzed, but we tested 161. We assigned most of these isolates to E. coli phylogroups B1 (52.8%) and A (36.6%), although phylogroups B2, C, D, E, and unknown also occurred. All isolates were assessed for the presence of several genes encoding virulence factors, such as adhesins (sfaDE, papC, afaBC III, ecpA, fimH, papA, and iha), toxins (hlyA, cnf1, sat, vat, and cdt), siderophores (iroN, irp2, iucD, ireA, and sitA), an invasion protein (ibeA), and serum resistance proteins (traT, KpsMTII, and ompT), and isolates from phylogroups B1, B2, and E showed up to 8 genes. Two isolates harbored the locus of enterocyte effacement (escN+) and lack the bundle-forming pilus (bfpB-) operon, which corresponds to a molecular profile of a subgroup of diarrheagenic E. coli (aEPEC), thus being classified as hybrid MPEC/aEPEC isolates. These isolates displayed a localized adherence-like pattern of adherence in HeLa cells and were able to promote F-actin polymerization underneath adherent bacteria. Based on the pulsed-field gel electrophoresis analyses, considerable genetic variability was observed. A low index of antimicrobial resistance was observed and 2 extended-spectrum β-lactamase-producing E. coli were identified, both harboring blaCTX-M15 gene, and were classified as ST10 and ST993 using multilocus sequence typing. A total of 148 (91.2%) isolates were weak biofilm producers or formed no biofilm. Because raw milk is still frequently consumed in Brazil, the occurrence of virulence factor-encoding genes from extraintestinal or diarrheagenic E. coli added to the presence of extended-spectrum β-lactamase-producing isolates can turn this veterinary medicine problem into a public health concern.

In-process real-time probiotic phenotypic strain identity tracking: The use of Fourier transform infrared spectroscopy.

Probiotic bacteria, capable of conferring benefits to the host, can present challenges in design, development, scale-up, manufacturing, commercialization, and life cycle management. Strain identification is one of the main quality parameters; nevertheless, this task can be challenging since established methodologies can lack resolution at the strain level for some microorganisms and\or are labor-intensive and time-consuming. Fourier transform infrared spectroscopy (FTIRS) has been largely used for the investigation of pathogenic species in the clinical field, whereas only recently has been proposed for the identification of probiotic strains. Within the probiotic industrial production, bacterial strains can be subjected to stressful conditions that may affect genomic and phenotypic characteristics; therefore, real-time monitoring of all the sequential growth steps is requested. Considering the fast, low-cost, and high-throughput features, FTIRS is an innovative and functional technology for typing probiotic strains from bench-top experiments to large-scale industrial production, allowing the monitoring of stability and identity of probiotic strains. In this study, the discriminatory power of FTIRS was assessed for four Lactiplantibacillus plantarum probiotic strains grown under different conditions, including temperatures (30 and 37°C) and medium (broth and agar), after consecutive sub-culturing steps. A comparison between the generated spectra with pulsed-field gel electrophoresis (PFGE) profiles was also performed. FTIRS was not only able to distinguish the strains of L. plantarum under different growth conditions but also to prove the phenotypic stability of L. plantarum type strain LP-CT after six growing steps. Regardless of the growth conditions, FTIRS spectra related to LP-CT constituted a unique hierarchical cluster, separated from the other L. plantarum strains. These results were confirmed by a PFGE analysis. In addition, based on FTIRS data, broth cultures demonstrated a higher reproducibility and discriminatory power with respect to agar ones. These results support the introduction of FTIRS in the probiotic industry, allowing for the step-by-step monitoring of massive microbial production while also guaranteeing the stability and purity of the probiotic strain. The proposed novel approach can constitute an impressive improvement in the probiotic manufacturing process.

Tandem Repeat of bla NDM-1 and Clonal Dissemination of a fosA3 and bla KPC-2 Co-Carrying IncR-F33: A-: B- Plasmid in Klebsiella pneumoniae Isolates Collected in a Southwest Hospital in China, 2010-2013.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been widespread in coastal cities of eastern China since 2009. However, how CRKP spreads and evolves in southwest China is unclear. We investigated the genetic characteristics and dissemination mechanisms of carbapenemase genes in forty-one non-repetitive CRKP isolates collected from a southwest hospital, Kunming, Yunnan, during 2010-2013. Drug susceptibilities were analyzed by using VITEK 2 compact system. Genetic relationships were ascertained based on multilocus sequence typing (MLST) and Pulsed-field gel electrophoresis (PFGE) analysis. Genetic backgrounds of bla KPC-2 and bla NDM-1 were revealed by DNA walking and high-throughput sequencing. All isolates were highly resistant to common antibiotics except for tigecycline. In total, 34 bla KPC-2, 3 bla NDM-1, 1 bla IMP-4 and 3 bla IMP-26 genes were identified and KP67 plasmid 1 co-harbored bla NDM-1 and bla IMP-26. Five sequence types, namely ST11, ST290, ST340, ST395 and ST437, were recognized by MLST. Surprisingly, bla KPC-2 was only detected in ST11 strains. We described a clonal dissemination of fosA3-positive IncR-IncF33:A-:B- multireplicon plasmid carrying the gene cassettes IS26-ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-korC-klcA-ΔrepB-Tn1721 in all ST11 isolates. Three bla NDM-1 positive isolates belonged to three different ST types and their bla NDM-1 genetic backgrounds were also distinct. Interestingly, the flanking regions of bla NDM-1 in KP67 and KP72 were duplicated into one to five copies in a form of tandem repeat by the transposition of IS91 like element. The bla NDM-1 of KP82 was carried on a common IncX3 plasmid. This study described the early epidemiological characteristics of bla KPC-2/bla NDM-1-carrying CRKP, and reported a new tandem repeat pattern of bla NDM-1 cluster in Yunnan. These findings extend our knowledge on the carbapenemase gene evolutions.

Genetic and Antimicrobial Resistance Profiles of Mammary Pathogenic E. coli (MPEC) Isolates from Bovine Clinical Mastitis.

Mammary pathogenic E. coli (MPEC) is one of the main pathogens of environmental origin responsible for causing clinical mastitis worldwide. Even though E. coli are strongly associated with transient or persistent mastitis and the economic impacts of this disease, the virulence factors involved in the pathogenesis of MPEC remain unknown. Our aim was to characterize 110 MPEC isolates obtained from the milk of cows with clinical mastitis, regarding the virulence factor-encoding genes present, adherence patterns on HeLa cells, and antimicrobial resistance profile. The MPEC isolates were classified mainly in phylogroups A (50.9%) and B1 (38.2%). None of the isolates harbored genes used for diarrheagenic E. coli classification, but 26 (23.6%) and 4 (3.6%) isolates produced the aggregative or diffuse adherence pattern, respectively. Among the 22 genes investigated, encoding virulence factors associated with extraintestinal pathogenic E. coli pathogenesis, fimH (93.6%) was the most frequent, followed by traT (77.3%) and ompT (68.2%). Pulsed-field gel electrophoresis analysis revealed six pulse-types with isolates obtained over time, thus indicating persistent intramammary infections. The genes encoding beta-lactamases detected were as follows: blaTEM (35/31.8%); blaCTX-M-2/blaCTX-M-8 (2/1.8%); blaCTX-M-15 and blaCMY-2 (1/0.9%); five isolates were classified as extended spectrum beta-lactamase (ESBL) producers. As far as we know, papA, shf, ireA, sat and blaCTX-M-8 were detected for the first time in MPEC. In summary, the genetic profile of the MPEC studied was highly heterogeneous, making it impossible to establish a common genetic profile useful for molecular MPEC classification. Moreover, the detection of ESBL-producing isolates is a serious public health concern.