Pulsed-field Gel Electrophoresis Techniques Research Articles

Outbreaks by Klebsiella oxytoca in neonatal intensive care units: Analysis of an outbreak in a tertiary hospital and systematic review

ObjectiveKlebsiella oxytoca can cause nosocomial infections, affecting vulnerable newborns. There are few studies describing nosocomial outbreaks in the neonatal intensive care units (NICU). In this study, a systematic review of the literature was carried out to know the main characteristics of these outbreaks and the evolution of one is described. MethodsWe conducted a systematic review in the Medline database up to July 2022, and present a descriptive study of an outbreak with 21 episodes in the NICU of a tertiary hospital, between September 2021 and January 2022. Results9 articles met the inclusion criteria. The duration of outbreaks was found to be variable, of which 4 (44.4%) lasted for a year or more. Colonization (69%) was more frequent than infections (31%) and the mortality rate was 22.4%. In studies describing sources, the most frequent was the environmental origin (57.1%). In our outbreak there were 15 colonizations and 6 infections. The infections were mild conjunctivitis without sequelae. Molecular typing analysis made it possible to detect 4 different clusters. ConclusionsThere is an important variability in the evolution and results of the published outbreaks, highlighting a greater number of colonized, use of PFGE (pulsed-field gel electrophoresis) techniques for molecular typing and implementation of control measures. Finally, we describe an outbreak in which 21 neonates were affected with mild infections, resolved without sequelae and whose control measures were effective.

Brotes por Klebsiella oxytoca en unidades de cuidados intensivos neonatales: análisis de un brote en un hospital de tercer nivel y revisión sistemática

ObjectivesKlebsiella oxytoca can cause nosocomial infections, affecting vulnerable newborns. There are few studies describing nosocomial outbreaks in the neonatal intensive care units (NICU). In this study, a systematic review of the literature was carried out to know the main characteristics of these outbreaks and the evolution of one is described. MethodsWe conducted a systematic review in the Medline database up to July 2022, and present a descriptive study of an outbreak with 21 episodes in the NICU of a tertiary hospital, between September 2021 and January 2022. ResultsNine articles met the inclusion criteria. The duration of outbreaks was found to be variable, of which 4 (44.4%) lasted for a year or more. Colonization (69%) was more frequent than infections (31%) and the mortality rate was 22.4%. In studies describing sources, the most frequent was the environmental origin (57.1%). In our outbreak there were 15 colonizations and 6 infections. The infections were mild conjunctivitis without sequelae. Molecular typing analysis made it possible to detect 4 different clusters. ConclusionsThere is an important variability in the evolution and results of the published outbreaks, highlighting a greater number of colonized, use of pulsed-field gel electrophoresis (PFGE) techniques for molecular typing and implementation of control measures. Finally, we describe an outbreak in which 21 neonates were affected with mild infections, resolved without sequelae and whose control measures were effective.

Study of the genetic diversity of Moraxella spp. isolates obtained from corneal abscesses

This is the first study of the genetic diversity of Moraxella spp. Isolates were detected in an Eye Hospital in the City of Buenos Aires. Due to the high frequency of Moraxella spp. observed in corneal abscesses, we decided to validate their identification at the species level, determine their drug susceptibility and perform molecular subtyping. Seventeen (17) isolates obtained from corneal abscesses were evaluated. The identification was carried out using a combination of biochemical tests and MALDI-TOF mass spectrometry. Of these isolates, 88.2% were identified as Moraxella lacunata, and 11.8% as Moraxella nonliquefaciens. Molecular subtyping was performed using the pulsed-field gel electrophoresis (PFGE) technique. All isolates were typable and thirteen digestion patterns were identified. Based on the obtained results, the PFGE technique using the SmaI enzyme can be used for epidemiological studies of strains of these species.

Identification of methicillin-resistant strains of Staphylococcus aureus isolated from humans and food sources by use mecA 1 and mecA 2 genes in Pulsed-field gel electrophoresis technique

Staphylococcus aureus causes mastitis in dairy cows, lambs, goats, and skin disorders in pigs and other animals. S. aureus causes localized purulent infections that affect soft tissues, bones, and other organs in humans. Using restriction patterns, the researchers want to isolate and identify methicillin-resistant Staphylococcus aureus (MRSA) strains from cattle and humans. They also hope to assess their genetic relatedness by comparing the mecA1 and mecA2 gene sequence discrepancies. Animals (223 strains) and people have been used to acquire S. aureus strains for study (83). The E-test was used to assess whether or not the bacteria were resistant to methicillin. The mecA1 and mecA2 genes were identified by using pulsed-field gel electrophoresis (PFGE) to analyze DNA restriction patterns. The results were shown. S. aureus strains from animals and men were resistant to methicillin in 32 (14.34 %) and 53 (63.8 %), respectively. PFGE was used to determine the differences between human and veterinary pathology strains. Two strains of bacteria collected from animals were discovered to be identical; nevertheless, microorganisms recovered from humans were found to be significantly similar to the bacteria recovered from animals. Both human and veterinary pathology were implicated in the development of methicillin resistance. The MRSA strains found in humans were much more significant than those found in animals. The strains recovered from animals exhibited a high degree of genetic heterogeneity. Still, the enormous number of indistinguishable bacteria in humans leads one to believe that a dominant clone is present. When it comes to the molecular characterization of MRSA isolates, PFGE might be regarded as the gold standard. Keywords. Animals, Human, MRSA, PFGE, Staphylococcus, mecA genes

A single-container procedure for multiple genomic DNA samples preparation for pulsed field gel electrophoresis

Genomic DNA preparation is a critical step for successful fingerprinting analysis by Pulsed Field Gel Electrophoresis (PFGE). This paper presents a simple and rapid protocol to prepare DNA samples from up to 24 bacterial isolates simultaneously. It involves performing the conventional PFGE sample preparation steps (cell growth and harvest, agarose-immobilization of intact cells, and DNA release and purification) into a 24-wells culture plate, without subjecting the biological material to repeated transference of containers. The single-container protocol rendered high quality genomic DNA from E. coli clinical isolates. The DNA yields obtained from samples prepared with the single-container protocol showed no differences to those obtained using the conventional DNA sample preparation for PFGE. This procedure is a cost-effective alternative that provides a larger capacity of analysis to the PFGE technique, which could be advantageous in a context of infectious disease outbreaks for pathogens molecular subtyping.

An efficient Pulsed-Field Gel Electrophoresis (PFGE) method for typing autolytic Lacticaseibacillus rhamnosus strains

Species of lactic acid bacteria, due to their versatile metabolism, are commonly used in food and feed products, both as technological starters and as health- and welfare-promoting agents. Correct strain identification in microbe-containing products is vital, and the Pulsed-Field Gel Electrophoresis (PFGE) typing method is considered the ‘gold standard’ for this purpose. This typing technique is widely used in molecular epidemiology, especially for the early detection of emerging isolates with food-safety implications, for outbreak surveillance, and for infection control. The autolytic behavior that we encountered when typing Lacticaseibacillus rhamnosus strains using the PFGE technique led us to modify the current method used for typing lactic acid bacteria. This study describes a PFGE method for the molecular typing of autolytic members of the lactic acid bacteria.•An efficient method for overcoming DNA degradation during PFGE analysis for typing Lacticaseibacillus rhamnosus strains is described.•The method described herein could be considered for typing autolytic lactic acid bacteria.

Diversity and antimicrobial resistance of Salmonella enterica serovars in surface river water and sediment

This study aimed to evaluate the contamination by Salmonella sp. in the Capinzal River, to determine the prevalent serovars, patterns of antimicrobial resistance, and the genetic relationships between the serovars identified. A total of 108 samples were collected from 2016 to 2018. The isolation of Salmonella spp. was conducted according to International Organization for Standardization (ISO) standards. The antimicrobial resistance profile of the Salmonella isolates was evaluated, and isolates were selected for serotyping and verification of genetic similarity using the Pulsed-Field Gel Electrophoresis (PFGE) Technique. Of the 108 samples collected, 35 (32.4%) were positive for Salmonella; 17.2% of the isolates were from the rural area; and 88.6% were from the urban area. Salmonella was isolated from all collect points along the river, with a higher incidence at the beginning of the urban area, indicating that contamination starts in the rural area and intensifies in the urban area of the city. A percentage of 35.1% of the Salmonella isolates were resistant to at least two antibiotics, while 18.9% were considered multidrug-resistant (resistant to at least two antibiotics of different classes). Seven serovars were distinguished from the serotyped isolates, with a prevalence rate of 23.5% for S. Infantis, S. Orion, and S. Javiana; 11.8% for S. Senfterberg, and 5.9% for S. Montevideo, S. Heidelberg, and S. enterica subsp. enterica (O: 6.8). The variability in specific restriction sites generated by PFGE resulted in 10 pulsotypes, separating mainly different serotypes.

Analysis of virulence genes and molecular typing of Listeria monocytogenes isolates from human, food, and livestock from 2008 to 2016 in Iran.

The frequency of Listeria monocytogenes isolates collected from a total of 1150 samples including food (n= 300), livestock (n= 50), and human clinical (n= 800) was evaluated during 2008-2016. Antimicrobial resistance patterns, virulence factors, and molecular characteristics of these isolates were analyzed using disk diffusion method, sequencing, serotyping, and pulsed-field gel electrophoresis (PFGE). The analysis of 44 L. monocytogenes isolates showed that 72.7% (32 of 44) of all the isolates belonged to Serotype 1/2c, and 15.9% (7 of 44) belonged to Serotype 3c. All 44 isolates were resistant to one or more antimicrobial agents with the most frequent resistance to penicillin (75%) and tetracycline (47.7%). Of the 44 L. monocytogenes strains, 100, 69.2, and 62.5% of livestock, human, and food strains were resistant to penicillin, respectively. Using pulsed-field gel electrophoresis (PFGE) technique, the isolates' genetic diversity was determined, and 28 PFGE patterns with 8 common (CT) and 20 single types (ST) were identified. This study highlights the high prevalence of Serotype 1/2c in clinical and livestock samples, while different serotypes were observed in food samples. The presence of rare serotypes such as 4c, belonging to the Lineage III, as well as 4e and 1/2c which are infrequent in Iran indicates that paying attention to uncommon serotypes, especially 1/2c, during the listeriosis outbreaks is necessary.

Detection of the agr System and Resistance to Antimicrobials in Biofilm-Producing S. epidermidis.

The ability of Staphylococcus epidermidis to produce virulence factors, such as biofilm, added to its increased resistance to antimicrobials can cause infections that are difficult to treat. Many staphylococcal virulence factors are under the control of the accessory gene regulator (agr). The objective of this study was to establish the agr locus and susceptibility of biofilm-producing S. epidermidis specimens to antimicrobial agents, through PCR reactions, reverse transcription polymerase chain reaction (RT-PCR), and the determination of minimum inhibitory concentration (MIC), and to analyze the clonal profile of 300 strains isolated from blood culture specimens from inpatients at a University Hospital in Brazil, over a 20-year period by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) techniques. The ica operon expression was shown in 83.6% strains, bhp gene in 11.5%, and aap gene in 32.8%. Oxacillin resistance was detected in 90.1%, while 4.9% showed tigecycline resistance, and intermediate resistance to quinupristin/dalfopristin was identified in 0.4%. Clonal profile determination showed 11 clusters, with the ST2 type determined as the major cluster. The S. epidermidis biofilm producer demonstrated a predominance of agr I locus, oxacillin resistance, and SCCmec III as well as the potential dissemination of pathogenic clones in hospital settings over long periods.

Genotypic diversity, and molecular and pathogenic characterization of Photobacterium damselae subsp. piscicida isolated from different fish species in Taiwan.

Photobacteriosis, caused by Photobacterium damselae subsp. piscicida (Phdp), is a serious disease in marine fish species worldwide. To date, the epidemiological characterization of this pathogen in Taiwan remains limited. In this study, we collected 39 Phdp isolates obtained from different farmed fish for phenotypic and genotypic analysis. Phenotype bioassays using API-20E and API-20NE systems showed that the Phdp is a homogeneous group. However, genotyping using the pulsed-field gel electrophoresis (PFGE) technique revealed genetic variability among Phdp isolates when 13 and 11 different PFGE band patterns were obtained with SmaI and NotI as restriction enzymes, respectively. Phylogenetic analysis using 16S rDNA and the Fur gene clustered Taiwanese isolates and other species of P. damselae in the same clade. In contrast, the ToxR phylogenetic tree, a powerful discriminatory marker, separated the two subspecies. Furthermore, the virulence-associated genes, AIP56, P55, PDP_0080, Sod and Irp1, were detected from all isolates. Virulence testing with nine representative isolates in cobia (Rachycentron canadum) and Asian sea bass (Lates calcarifer) showed that some were highly pathogenic with 80%-100% mortality rates. This study provides epidemiological data of Phdp infections in farmed fish in Taiwan, which is necessary to develop comprehensive prevention and control strategies for the disease.

E. coli diversity: low in colorectal cancer

BackgroundEscherichia coli are mostly commensals but also contain pathogenic lineages. It is largely unclear whether the commensal E. coli as the potential origins of pathogenic lineages may consist of monophyletic or polyphyletic populations, elucidation of which is expected to lead to novel insights into the associations of E. coli diversity with human health and diseases.MethodsUsing genomic sequencing and pulsed field gel electrophoresis (PFGE) techniques, we analyzed E. coli from the intestinal microbiota of three groups of healthy individuals, including preschool children, university students, and seniors of a longevity village, as well as colorectal cancer (CRC) patients, to probe the commensal E. coli populations for their diversity.ResultsWe delineated the 2280 fresh E. coli isolates from 185 subjects into distinct genome types (genotypes) by PFGE. The genomic diversity of the sampled E. coli populations was so high that a given subject may have multiple genotypes of E. coli, with the general diversity within a host going up from preschool children through university students to seniors. Compared to the healthy subjects, the CRC patients had the lowest diversity level among their E. coli isolates. Notably, E. coli isolates from CRC patients could suppress the growth of E. coli bacteria isolated from healthy controls under nutrient-limited culture conditions.ConclusionsThe coexistence of multiple E. coli lineages in a host may help create and maintain a microbial environment that is beneficial to the host. As such, the low diversity of E. coli bacteria may be associated with unhealthy microenvironment in the intestine and hence facilitate the pathogenesis of diseases such as CRC.

Characterization of Staphylococcus aureus strains and evidence for the involvement of non-classical enterotoxin genes in food poisoning outbreaks.

To an increasing extent, molecular and genetic characterization is now used to investigate foodborne outbreaks. The aim of this study was to seek molecular links among coagulase-positive staphylococci (CPS) isolated from three recent food poisoning outbreaks in Romania using polymerase chain reaction and pulsed-field gel electrophoresis techniques. The 19 CPS isolates were identified as Staphylococcus aureus by detection of the 23S rDNA gene. Among them, 15 carried at least one staphylococcal enterotoxin-encoding gene. The Calaraşi outbreak strains grouped in pulsotype 2 and were sed/sej/ser-positive, whereas the Arad outbreak strains clustered in pulsotype 17 and were either sed/seg/sei/sej/ser- or seg/sei-positive. The Piteşti outbreak strains clustered in pulsotype 1 and, surprisingly, possessed only one enterotoxin gene, i.e. seh. Similar to other European countries, the seh gene has been identified with increasing frequency in Romanian outbreaks; this highlights the importance of considering the application of methods recommended for staphylococcal enterotoxin regulation in Europe.

Detection of common clones of Salmonella enterica serotype Infantis from human sources in Tehran hospitals.

The aims of this study were to investigate antibiotic resistance pattern and molecular characterization of Salmonella Infantis strains, isolated from human sources in Tehran hospitals from 2008 to 2010. Infection caused by Salmonella is one of the major public health problems. Despite the clinical importance of Salmonella enteric subsp. enteric serovar Infantis in humans, there is no information available about the common clones of Salmonella Infantis in clinical isolates in Iran. S. Infantis strains were identified by conventional microbiological and serological testing. The antimicrobial susceptibility of the S.Infantis isolates was determined using the disk diffusion method. The genetic relatedness and the dominant clones of S. Infantis strains were detected by the Multi Locus Sequence Typing (MLST) and pulsed-field gel electrophoresis (PFGE) techniques. More than 80% of the S. Infantis isolates represented multidrug-resistant patterns. PFGE revealed high genetic similarity among S. Infantis strains. While, MLST indicated high-clonal similarity among strains, where all S. Infantis strains were assigned to the ST32 sequence type. This is the first study in Iran conducted to determine the sequence types of S. Infantis in clinical isolates using MLST. The genetically closed MDR S. Infantis clones were responsible for the apparent endemic occurrence of salmonellosis, caused by this Salmonella serovar, in Tehran.

Genotypic characterization of Escherichia coli strains isolated from dairy cattle environment

The aim of this study was to characterize the diversity of Escherichia coli strains involved in the dispersion of virulence genes. 152 E. coli strains originated from dairy cattle environment were evaluated through phenotypic and proteomic assays. These samples were investigated for the presence of virulence genes (eaeA, stxI, stxII, ST, LT, eagg, ial) and biofilm related genes (fimH, csgA, flu). Eighteen profiles were obtained and 30 isolates were selected for macrorestriction assay by Pulsed-Field Gel Electrophoresis (PFGE) technique. PFGE patterns of XbaI-digested performed to determine the clonal relatedness of E coli isolates. A total of 27 pulsotypes of E. coli were identified with a low percentage of genetic similarity (≤68%) demonstrating a high genetic diversity in the isolates tested. Furthermore, the presence of biofilm-associated related gene (fimH) can contribute to the dispersion and persistence of this pathogen into the milk environment. Also, all of the pulsotypes (n = 8) with genetic similarity from 95% were present in milk samples, feces and liner were found in different seasons of the year, sharing at least one gene associated with biofilm formation. So the characterization of the genetic diversity of E. coli is important for understanding its dispersion in order to assist in the implementation of measures to prevent the spread of these agents in milk production environment. Key words: circulating clones, diversity, Escherichia coli, persistence, Pulsed-Field Gel Electrophoresis (PFGE), virulence gene.

Genetic diversity reflects geographical origin of Ralstonia solanacearum strains isolated from plant and water sources in Spain.

The characterization and intraspecific diversity of a collection of 45 Ralstonia solanacearum strains isolated in Spain from different sources and geographical origins is reported. To test the influence of the site and the host on strain diversity, phenotypic and genotypic analysis were performed by a polyphasic approach. Biochemical and metabolic profiles were compared. Serological relationship was evaluated by Indirect-ELISA using polyclonal and monoclonal antibodies. For genotypic analysis, hrpB and egl DNA sequence analysis, repetitive sequences (rep-PCR), amplified fragment length polymorphism (AFLP) profiles and macrorestriction with XbaI followed by pulsed field gel electrophoresis (PFGE) were performed. The biochemical and metabolic characterization, serological tests, rep-PCR typing and phylogenetic analysis showed that all analysed strains belonged to phylotype II sequevar 1 and shared homogeneous profiles. However, interesting differences among strains were found by AFLP and macrorestriction with XbaI followed by PFGE techniques, some profiles being related to the geographical origin of the strains. Diversity results obtained offer new insights into the biogeography of this quarantine organism and its possible sources and reservoirs in Spain and Mediterranean countries.

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.

Clinical Isolates of Acinetobacter baumannii From Tehran Hospitals: Pulsed-field Gel Electrophoresis Characterization, Clonal Lineages, Antibiotic Susceptibility, and Biofilm-forming Ability

Background: Acinetobacter baumannii is known as a potential pathogen in hospitals and is responsible for the dramatic increase in carbapenem resistance in Iran in the recent years. Objectives: The current study aimed at determining the genetic association of the isolates by the pulsed-field gel electrophoresis (PFGE) technique, identify international clones, evaluate biofilm formation ability and its relationship with antibiotic resistance. Methods: In the current study, a total of 48 A. baumannii isolates were collected from 2 hospitals in Tehran, Iran, from 2010 to 2012. Isolates were subjected to antimicrobial susceptibility testing, determination of carbapenemase encoding genes, biofilm formation, and genetic relationships analysis. Results: The obtained results demonstrated that the rate of resistance to carbapenem, meropenem, imipenem, and doripenem was 76%. The carbapenemase-encoding gene bla(OXA-23-like) was found in 32 isolates, while bla(OXA-40-like) (bla(OXA-24-like)), bla(OXA-58-like), bla(VIM-type) and bla(IMP-type) were found in 11, 1, 19. and 5 isolates, respectively. When the lineage of the isolates was evaluated by the multiplex polymerase chain reaction (PCR), it was found that 28 isolates belonged to group 1 and 8 isolates to group 2. None of the isolates belonged to group 3. Twelve isolates could not be typed by this method. The study findings interestingly demonstrated that 13 isolates showed no biofilm formation. Data of biofilm formation also demonstrated that 28, 4, and 3 remaining isolates had weak, moderate, and strong biofilm formation, respectively. The pulsed field gel electrophoresis result revealed 11 unique clones. Conclusions: International clone 1 was the most commonly identified clone in the current study. This clone was mostly associated with bla(OXA-23-like) gene; therefore, 64% of the isolates in this clone possessed bla(OXA-23-like) gene. Keywords Author Keywords:Acinetobacter baumannii; Electrophoresis; Gel; Pulsed-Field; Biofilm

Molecular Typing of Brucella melitensis Isolated from Patients and Animals by Pulsed Field Gel Electrophoresis from Iran

Background: The Brucellosis is recognized as a significant public health problem, with major economic and financial burdens in countries where the disease remains endemic. The disease is one of the most important worldwide zoonosis affecting livestock and humans. The aim of the present study was to genotypically characterize Brucella strains isolated from human and animal samples in two provinces of Iran. Methods: Twenty seven Brucella strains isolated from patients and animals during April 2015 - December 2016. Thirteen human and fourteen animal strains were identified by biochemical tests and confirmed by amplification of fragment 1100bp omp2a gene using Polymerase Chain Reaction (PCR) method. Isolates were genotypically characterized by XbaI digestion and pulsed-field gel electrophoresis (PFGE) technique. Results: In PFGE analysis totally, 7 common clones and 3 single clones were obtained. In genotyping, in two clones/clusters, human isolates were grouped with genotypes from animal isolates. Also PFGE results indicated 21 Brucella strains an overall similarity higher than 90, and three clusters based on 100 similarity were revealed. Conclusion: According to dendrogram clinical strains had a high degree of homology compared to animal strains. The information of this study indicating a close genetic relatedness or common origin of the isolates in the two geographical region of Iran and it implies that B. melitensis cross-infected between human and livestock.

Molecular Characterization of Multidrug Resistant Strains of Acinetobacter baumannii Isolated from Intensive Care Units in West of Iran

According to the results of various studies using phenotypic methods, the prevalence of Multidrug Resistant (MDR) Acinetobacter baumannii (A. baumannii) isolates has been increasing worldwide. Pulsed-Field Gel Electrophoresis (PFGE) technique is known as the gold standard method to determine clonal characterization of bacterial species, especially A. baumannii. To determine the clonal relatedness and investigate the prevalence of integron classes 1 and 2 and genes encoding OXA-23 and 24 in A.baumanii isolates. A cross-sectional study was conducted from November 2011 to January 2013. A total of 140 A.baumannii isolates collected from three hospitals of Kermanshah were considered out of which 75 ICU isolates were included in this study. Antibiotics susceptibility test was done by disk diffusion method. Polymerase Chain Reaction (PCR) was performed in order to detect class 1 and 2 integrons and blaOXA-23-like, blaOXA-24-like genes. Isolates identified as MDR from a total of 75 Intensive Care Units (ICU) strains were subjected to genotyping for clonal relatedness. A total of 37 isolates among 75 ICU isolates were identified as MDR. The maximum drug resistance was observed against ceftriaxone, mezlocycline, cefotaxime, piperacilin, ciprofloxacin and imipenem. Frequency of Class 1 and Class 2 Integrons, blaOXA-23-like and blaOXA-24-like genes were 33(44%), 27(36%), 60(80%) and 14(18.6%) respectively. Four clusters with high level of similarity were obtained showing homogeneity among MDR isolates. Significant correlation between presence of integrons and resistance to different classes of antibiotic was observed in this study. Monitoring of drug resistance using gene integrase PCR and blaOXA gene by cluster analysis is very important to plan specific infection control measures due to MDR A. baumannii.

Mesophilic Lactic Acid Bacteria Diversity Encountered in Brazilian Farms Producing Milk with Particular Interest in Lactococcus lactis Strains.

The milk produced in regions with different traditions in Brazil is used for artisanal product production, which is characterized by different sensorial characteristics. This study aimed to identify the bacterial ecosystem of farms located in a traditional dairy region in the state of Minas Gerais and to characterize Lactococcus lactis strains, the species of interest in this study, using a multilocus sequence typing (MLST) protocol and pulsed-field gel electrophoresis (PFGE) technique. Samples were collected from raw milk and dairy environment from six farms. A total of 50 isolates were analyzed using 16S rRNA sequencing and species-specific PCR. Five genera were identified: Lactobacillus, Leuconostoc, Lactococcus, Enterococcus, and Staphylococcus, from ten different species. MLST (with six housekeeping genes) and PFGE (with SmaI endonuclease) were used for the characterization of 20 isolates of Lactococcus lactis from a dairy collection in this study. Both methods revealed a high clonal diversity of strains with a higher discriminatory level for PFGE (15 pulsotypes), compared to MLST (12 ST). This study contributes to the preservation of the Brazilian dairy heritage and provides insights into a part of the LAB population found in raw milk and dairy environment.