Pulsed-field Gel Electrophoresis Groups Research Articles

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.

Genotypic correlation between Salmonella Enteritidis isolates from broiler breeders and hatchery flocks

Abstract In this study, Salmonella Enteritidis strains isolated from dust and environmental materials from different flocks located in Turkey’s Western Black Sea region were examined by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 59 S. Enteritidis strains isolated from broiler breeder and hatchery flocks, and one S. Enteritidis strain isolated from a stool sample of a farm worker were examined. PFGE analysis revealed two major PFGE groups and nine different macro restriction profiles. It was determined that 85% (51/60) of the strains were close to each other and comprised Group I. All S. Enteritidis strains had the same sequence type (ST): ST11. Isolation of strains with a single genotype suggests that there may be a cross transmission between the flocks.

Prevalence, Characterization, and Antimicrobial Susceptibility of Listeria monocytogenes from Raw Beef and Slaughterhouse Environments in Korea.

The prevalence of Listeria monocytogenes in raw beef and in slaughterhouse environments was investigated from April 2019 to February 2020. Three hundred raw beef samples were purchased from 50 retailers and 10 restaurants (5 samples per source). One hundred and thirty-four samples from slaughterhouse environments were collected by swabbing (10 × 10 cm) the surfaces, gloves, splitting saw, and drains. L. monocytogenes was detected and identified according to the method described in ISO 11290-1, and confirmed by 16S rRNA sequencing. L. monocytogenes was detected in raw beef (2/300, 0.7%), gloves used in carcass splitting (6/21, 28.6%), the splitting saw (1/18, 5.6%), and the drain zone (1/15, 6.7%). All isolates were serotype 1/2a or 1/2c, based on screening using multiplex PCR-based serogrouping assay and serotyping kit for O-H antigens. Pulsed-field gel electrophoresis (PFGE) following ApaI digestion of eight PFGE pulsotypes and four PFGE groups were identified. Biofilm formation analysis using Crystal Violet staining revealed the highest biofilm formation in strain LM-16, followed by D190613. Although L. monocytogenes isolates were susceptible to most antimicrobials, some resistance to penicillin (8/15, 53.3%) and tetracycline (2/15, 13.3%) was observed. Through PFGE, G190426, G190829, and G200210 isolated from the same location in this study were genetically homologous similar to the LM-16 strain, previously isolated from beef carcass in 2006. These results suggest that LM-16 has been continuously present in biofilms in the slaughterhouse environments since 2006. Our study indicates that L. monocytogenes contamination in raw beef could consistently occur during beef processing in slaughterhouse environments through contact with gloves, splitting saws, and drains.

Quick identification and epidemiological characterization of Francisella tularensis by MALDI-TOF mass spectrometry

IntroductionCurrently, Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry (MALDI-TOF MS) is being evaluated for its efficacy as a fast bacterial typing tool due to its great speed compared to other molecular methods. In this study, we evaluated MALDI-TOF as a tool for quick identification and typing of Francisella tularensis. Materials and methodsThis study encompassed 86 strains from two different geographical origins (Spain and the Czech Republic), which were previously characterised by Pulsed-Field Gel Electrophoresis (PFGE) and Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA). The direct colony method was used for microbial identification. High-quality spectra of the 86 strains were obtained and their main spectra profiles (MSPs) were created for epidemiological typing using MALDI-TOF. Based on the MSPs, principal components were generated and a dendrogram was constructed. An in-house MALDI-TOF library entry was created for each group of PFGE and MLVA strains based on their high-quality spectra. Two dendrograms were obtained using these entries and the unique peaks in each entry were searched. ResultsAll strains were correctly identified to the species level. No clear divisions were found in the 86-strain dendrogram; however, Spanish and Czech strains appeared separately in dendrograms created using MLVA and PFGE entries. Entries from our in-house MALDI-TOF library revealed 2–4 biomarker peaks for the detection of the five PFGE groups and 1–12 biomarker peaks for the detection of the seven MLVA groups. Finally, two and one specific biomarkers were found in the Czech and Spanish strains, respectively. ConclusionMALDI-TOF can be used to accurately identify F. tularensis strains in less than 15 min. Moreover, data on geographical origin and PFGE and MLVA groups could be obtained in less than one hour after colony growing.

Listeria monocytogenes in sliced cheese and ham from retail markets in southern Brazil.

The aims of this study were to evaluate the occurrence of Listeria monocytogenes and Salmonella spp. in sliced cheese and ham from retail markets in southern Brazil, as well as to perform molecular characterization and to assess the antimicrobial resistance profile of the isolates. Samples (n=160) of sliced cheese and ham were collected at retail level from the city of Pelotas, Brazil. The isolation of L. monocytogenes and Salmonella spp. was performed and the isolates were confirmed by PCR, submitted to antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). Listeria monocytogenes was found in 9.4% (15/160) of the samples. All L. monocytogenes isolates were positive for the prs, inlA, inlC and inlJ genes. Salmonella spp. was not isolated. Regarding the antimicrobial susceptibility, one (6.6%) L. monocytogenes isolate was resistant to streptomycin and four (26.6%) to clindamycin. Macrorestriction analysis with ApaI and AscI enzymes yielded two major PFGE groups I and II. All L. monocytogenes isolates showed virulence genes, and some of them were resistant to clinically used antimicrobials, representing a risk to public health. Moreover, PFGE patterns with high similarity were visualized in L. monocytogenes isolates at different times, demonstrating adaptability of the pathogen at retail level in the region.

Strain variation and antigenic divergence among Bordetella pertussis circulating strains isolated from patients in Iran.

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

High Diversity of Antimicrobial Resistance Genes, Class 1 Integrons, and Genotypes of Multidrug-Resistant Escherichia coli in Beef Carcasses.

Multidrug-resistant Escherichia coli can contaminate food meat during processing and cause human infection. Phenotypic and genotypic characterization of the antimicrobial resistance were conducted for 45 multidrug-resistant E. coli isolates from 208 samples of beef carcasses. The mechanisms of resistance were evaluated using polymerase chain reaction and sequencing methods, and the clonal relationship among isolates was evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Different variants of bla, tet, flo, dfrA, and aadA genes were detected in most of the strains resistant to β-lactam, tetracycline, chloramphenicol, sulfonamides, and aminoglycosides, respectively. Extended-spectrum β-lactamase (ESBL)-producing E. coli was found in 42.2% of the 45 E. coli isolates and the most commonly detected ESBL genotypes were CTX-M group 1 and 9. Class 1 integrons with nine different arrangements of gene cassettes were present in 28 of 45 E. coli isolates. Twenty-nine PFGE groups and 24 MLST types were identified in their clonal structure. This study revealed that E. coli isolates from beef contained high diversity of antimicrobial resistance genes, integrons, and genotypes. These results highlighted the role of beef meat as a potential source for multidrug-resistant E. coli strains and the need for controlling beef safety.

Characterization of Salmonella spp. from wastewater used for food production in Morogoro, Tanzania.

Wastewater use for crop irrigation and aquaculture is commonly practiced by communities situated close to wastewater treatment ponds. The objective of this study was to characterize Salmonella spp. and their antimicrobial susceptibility patterns among isolates from wastewater and Tilapia fish. A total of 123 Salmonella spp. isolates were isolated from 52 water and 21 fish intestinal samples. Genotyping of Salmonella spp. isolates was done by Pulsed-field Gel Electrophoresis (PFGE). Antimicrobial susceptibility testing was done by the minimal inhibitory concentration (MIC) technique. A total of 123 Salmonella spp. isolates represented 13 different serovars and 22 PFGE groups. Salmonella serovars showed resistance to 8 out of 14 antimicrobials; sulfamethaxazole (94%), streptomycin (61%), tetracycline (22%), ciprofloxacin and nalidixic acid (17%), trimethoprim (11%); gentamycin and chloramphenicol (6%). Salmonella Kentucky, S. Chandans, S. Durban and S. Kiambu showed multiple antimicrobial resistance to 7, 6 and 3 antimicrobials, respectively. This study has demonstrated that wastewater at the study sites is contaminated with Salmonella spp. which are resistant to common antimicrobials used for treatment of diseases in humans. Wastewater may, therefore, contaminate pristine surface water bodies and foodstuffs including fish and irrigated crops as well as food handlers.

Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb.

Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958-2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and Ö-6 to Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advantageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.

Molecular epidemiology and spatiotemporal analysis of hospital-acquired Acinetobacter baumannii infection in a tertiary care hospital in southern Thailand

Acinetobacter baumannii is a major hospital-acquired pathogen in Thailand that has a negative effect on patient survival. The nature of its transmission is poorly understood. To investigate the genotypic and spatiotemporal pattern of A.baumannii infection at a hospital in Thailand. The medical records of patients infected with A.baumannii at an 800-bed tertiary care hospital in southern Thailand between January 2010 and December 2011 were reviewed retrospectively. A.baumannii was identified at the genomospecies level. Carbapenemase genes were identified among carbapenem-resistant isolates associated with A.baumannii infection. A spatiotemporal analysis was performed by admission ward, time of infection and pulsed-field gel electrophoresis (PFGE) groups of A.baumannii. Nine PFGE groups were identified among the 197 A.baumannii infections. All A.baumannii isolates were assigned to International Clonal Lineage II. blaOXA-23 was the most prevalent carbapenemase gene. Outbreaks were observed mainly in respiratory and intensive care units. The association between PFGE group and hospital unit was significant. Spatiotemporal analysis identified 20 clusters of single PFGE group infections. Approximately half of the clusters involved multiple hospital units simultaneously. A.baumannii transmitted both within and between hospital wards. Better understanding and control of the transmission of A.baumannii are needed.

The prevalence, genetic diversity and antibiotic resistance of Staphylococcus aureus in milk, whey, and cheese from artisan farm dairies

In this study, coagulase positive staphylococci were detected in 45% of the 69 bovine milk, whey and cheese samples taken from five farm dairies, and all raw milk samples were contaminated. Genetic diversity, staphylococcal enterotoxin genes and antimicrobial susceptibility in putative Staphylococcus aureus isolates were investigated. Sixty-one percent of the 72 isolates analysed belonged to the same pulsed field gel electrophoresis group. The spa-typing revealed seven different spa types, t2678 being the most prevalent, but t127 and t197 were also detected. Sixteen different toxin gene profiles were identified in 87.5% of the isolates with sec and tst being the most frequent (52.5%), followed by seg and seh. All isolates were methicillin-sensitive S. aureus, and sensitive to the 12 antibiotics tested. The prevalence of S. aureus and the high diversity of isolates carrying enterotoxin genes constitute grounds for food safety concern in artisanal cheese making, whether pasteurised or not.

Molecular and Phenotypic Characterization Revealed High Prevalence of Multidrug-Resistant Methicillin-Susceptible Staphylococcus aureus in Chongqing, Southwestern China.

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for ∼40% of staphylococcal infections in China. However, the molecular characterization of MSSA is not well described. In this study, 124 MSSA strains collected in 2013 from a comprehensive teaching hospital in Chongqing, Southwestern China, were subjected to antibiotics susceptibility testing and molecular typing, including multilocus sequence typing, staphylococcal protein A (spa) gene typing, accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) typing, Panton-Valentine leukocidin (pvl) gene detection, and antibiotic-resistant gene detection. MSSA strains exhibited high genetic heterogeneity. A total of 10 PFGE groups, 26 sequence types, and 47 spa types were identified. Type I (62.9%) was the most frequent agr type, followed by type II (15.3%), type IV (11.3%), and type III (10.5%). The prevalence of pvl genes was 27.4% (34/124). Notably, 44.4% (55/124) of MSSA strains were multidrug resistance (MDR), and MDR isolates were mostly resistant to penicillin, erythromycin, and clindamycin. The resistance gene blaZ was present in 84.7% of strains, ermC was present in 85.5% of strains, ermA was present in 28.2% of strains, tetK was present in 16.1% of strains, tetM was present in 6.5% of strains, and aacA-aphD was present in 2.6% of strains. These data demonstrated the high prevalence of MDR MSSA in Chongqing, thereby indicating the need to control MSSA infection.

Comparison of Mannheimia haemolytica isolates from an outbreak of bovine respiratory disease

The objective of this study was to determine the clonal relatedness of Mannheimia haemolytica isolates responsible for an outbreak of bovine respiratory disease in a commercial feedlot. The isolates were obtained from the lungs of 21 calves with fatal pneumonia that were part of a group of 206 total calves. All isolates were serotyped and analyzed by pulsed-field gel electrophoresis (PFGE) and for antibiotic sensitivity patterns. ELISA and immunoblotting assays were performed to compare serum antibody levels to M. haemolytica antigens in calves with fatal pneumonia to those calves that survived the outbreak. Isolates were categorized into 14 different PFGE groups based on 90% similarity. Two Group D isolates (1 and 6), and 3 Group H isolates (14, 15, and 16) were characterized as 100% similar. Antimicrobial susceptibility profiles defined 8 groups based on differences in patterns of resistance between isolates. The two 100% similar isolates from PFGE Group D were both in susceptibility Group 1. All but isolate 14 from PFGE Group H (3, 15, 16, and 19) were in susceptibility Group 4a. Serum antibody levels to M. haemolytica antigens in the dead calves were not different than the antibody levels in the 185 calves that survived the outbreak. Immunoblots of selected isolates from each of the PFGE groups demonstrated only minimal differences in antigenic profiles between strains when reacted with serum from calves that either died from or survived the outbreak. Based on the characteristics of these isolates, multiple strains of M. haemolytica were responsible for fatal pneumonia during this outbreak.

Temporal trends and clonal diversity of penicillin non-susceptible pneumococci from meningitis cases from 1996 to 2012, in Salvador, Brazil.

BackgroundHospital-based surveillance for pneumococcal meningitis has been conducted since January 1996 in the city of Salvador, Brazil. The purpose of this study was to describe the temporal evolution of Penicillin Non-Susceptible Streptococcus pneumoniae (PNSSP) in regards to serotype distributions and clonal diversity recovered from meningitis cases over 17 years.MethodsBroth microdilution was used to identify pneumococcal isolates that were PNSSP (Minimum Inhibitory Concentration > 0.12 μg/ml). The annual incidence rate of meningitis cases was calculated. Serotyping was defined using multiplex polymerase chain reaction assays and quellung reaction. Genetic diversity of PNSSP isolates was assessed using both pulsed-field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) analyses.ResultsA total of 854cerebrospinal fluid (CSF) culture pneumococcal isolates were tested by broth microdilution method and serotyped. A total of 173 (20.3 %) were penicillin non-susceptible (PNSSP) (Minimum Inhibitory concentration ≥ 0.12 μg/ml). The annual incidence of meningitis cases declined from 1.65/100,000 population (1996) to 0.2/100,000 population in 2012 and the rate due to PNSSP declined 82 % over the 17-years of surveillance. PNSSP isolates were restricted to 13 serotypes, being the most common ones serotypes14 (45.1 %; 78/173), 23 F (19.1 %; 33/173), 6B (14.4 %; 25/173), 19 F (9.2 %; 16/173) and 19A (5.2 %; 9/173). Among the PNSSP isolates, 94 % had serotypes represented in the 10-valent conjugate vaccine (PCV10). The predominant serotype 14 clonal groups were identified as PFGE group A/multilocus sequence type 66 (ST66) [35.3 % (61/173)] and PFGE group GK/ST156 [4.6 % (8/173)], the latter one associated with high level resistance to penicillin and ceftriaxone.ConclusionsOur results show sustained reductions in pneumococcal meningitis cases in the Metropolitan region of Salvador from 1996 to 2012. This might reflect a beneficial impact of conjugate vaccines. Continued surveillance and further studies need to be conducted to better understanding on PCV10 vaccine impact.

Dissemination of multiple carbapenem-resistant clones of Acinetobacter baumannii in the Eastern District of Saudi Arabia.

It has previously been shown that carbapenem-resistant Acinetobacter baumannii are frequently detected in Saudi Arabia. The present study aimed to identify the epidemiology and distribution of antibiotic resistance determinants in these bacteria. A total of 83 A. baumannii isolates were typed by pulsed-field gel electrophoresis (PFGE), and screened by PCR for carbapenemase genes and insertion sequences. Antibiotic sensitivity to imipenem, meropenem, tigecycline, and colistin were determined. Eight different PFGE groups were identified, and were spread across multiple hospitals. Many of the PFGE groups contained isolates belonging to World-wide clone 2. Carbapenem resistance or intermediate resistance was detected in 69% of isolates. The blaVIM gene was detected in 94% of isolates, while blaOXA–23–like genes were detected in 58%. The data demonstrate the co-existence and wide distribution of a number of clones of carbapenem-resistant A. baumannii carrying multiple carbapenem-resistance determinants within hospitals in the Eastern Region of Saudi Arabia.

Comparison of Whole-Genome Sequencing and Molecular-Epidemiological Techniques for Clostridium difficile Strain Typing.

We analyzed in parallel 27 pediatric Clostridium difficile isolates by repetitive sequence-based polymerase chain reaction (RepPCR), pulsed-field gel electrophoresis (PFGE), and whole-genome next-generation sequencing. Next-generation sequencing distinguished 3 groups of isolates that were indistinguishable by RepPCR and 1 isolate that clustered in the same PFGE group as other isolates.

Investigation of the molecular epidemiology of Acinetobacter baumannii isolated from patients and environmental contamination.

The objective of this work was to investigate correlations between Acinetobacter baumannii isolates from neurosurgical intensive care unit patients and its environment. This is a prospective, observational study. The minimal inhibitory concentrations of antimicrobial agents against 27 clinical and 28 environmental isolates were determined by the agar dilution method. Molecular genotyping was performed by enterobacterial repetitive intergenic consensus PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of carbapenemase and metallo-β-lactamase genes were analyzed by specific PCRs and DNA sequencing. From the clinical A. baumannii isolates, 25.9% were found resistant to minocycline, 51.9% to cefoperazone-sulbactam, 59.3% to imipenem and 70% resistant to other antimicrobial agents. Environmental isolates were more sensitive compared with clinical isolates (P<0.05). Twenty-seven clinical isolates comprised three ERIC-PCR genotypes, four major PFGE pulsotypes and five distinct MLST sequence types (STs) (ST208, ST368, ST191, ST195, ST540), all belonging to CC92 with only one locus (gpi) difference among them. Twenty-eight environmental isolates showed more diverse genetic types than clinical isolates and comprised six ERIC-PCR groups, nine PFGE groups and two main STs (ST208, ST229). Four clinical and 15 environmental isolates could not be identified by MLST and were assigned to non-clonal STs. We identified the presence of the blaOXA-23 carbapenemase encoding gene in most of the clinical (21/27) but fewer in the environmental isolates (3/28). The A. baumannii strains isolated from patients were genetically similar to the environmental strains, with CC92 members as the major fraction but with different antibiotic susceptibilities.

Division of HumanListeria monocytogenesPulsed-Field Gel Electrophoresis (PFGE) Types Belonging to Lineage I (Serovar 4b, 1/2b, and 3b) into PFGE Groups

The 63 pulsed-field gel electrophoresis (PFGE) types identified among 427 clinical isolates of Listeria monocytogenes that were characterized in a previous study by serotyping and PFGE (AscI) could be further divided into 17 PFGE groups. While the 63 PFGE types, all part of lineage I, were established based on the number and distribution of all bands in each DNA profile, the 17 PFGE groups were based on the configuration of small bands with sizes <145.5 kb. The 30 PFGE types of L. monocytogenes serovar 4b isolates (n=334) were divided into 8 PFGE groups; the 32 PFGE types of serovar 1/2b isolates (n=90) and the serovar 3b isolates (n=3, 1 PFGE type) were divided into 9 PFGE groups. An association was observed between PFGE groups and serovars. L. monocytogenes isolates belonging to PFGE groups I, J, Q, R, X, Z, Ö-4, and Ö-5 all shared serovar 4b, whereas isolates from PFGE groups D, G, O, P, T, U, Ö-1, Ö-2, and Ö-3 shared serovar 1/2b. Small fragments <33.3 kb were nonvisible in all L. monocytogenes isolates. From the results of the present study, a procedure for accelerating the identification of PFGE types when analyzing new PFGE profiles can be suggested. Therefore, we propose a stepwise procedure to PFGE profiling by first identifying the PFGE group using the smaller band patterns <145.5 kb, and then determining PFGE types based on the band patterns >145.5 kb.

Drug resistance and virulence of uropathogenic Escherichia coli from Shanghai, China.

Uropathogenic Escherichia coli (UPEC) is the major cause of urinary tract infections (UTIs). In the present study, 198 E. coli isolates from patients with UTIs in Shanghai in 2008 were examined by susceptibility testing, with an extremely high number (153/198) showing multidrug resistance (MDR). And, the expression of extended-spectrum β-lactamases (ESBLs) reached 48.5% (96/198). The resistance rates to penicillins, fluoroquinolone, folate pathway inhibitors and first- and second-generation cephalosporins were high. Molecular analyses showed that the CTX-M-9 group (70/96) was the most common CTX-M group among UPEC, followed by the CTX-M-1 group (27/96). Phylogenetic group D accounted for 42.4% (84/198) of the isolates, exhibiting the highest ESBLs (50/84) and MDR (75/84) rates. Virulence genes were present in a significantly high proportion in the phylogenetic group B2 isolates, except for the afaBC gene. The ESBL-producing strains analyzed by pulsed-field gel electrophoresis (PFGE) were clustered into six groups at a cutoff of 67%. Notably, the findings that afaBC was specific to phylogenetic group D and PFGE group I and was correlated with the CTX-M-9 group were different from a previous report. In conclusion, knowledge of antimicrobial resistance data and virulence factors may enable clinicians to tailor empirical antibiotic treatments for UTIs.

Spread of extended spectrum cephalosporinase-producing Escherichia coli clones and plasmids from parent animals to broilers and to broiler meat in a production without use of cephalosporins.

This study investigated the occurrence of extended spectrum cephalosporinase (ESC)-producing Escherichia coli in a broiler production with no cephalosporin use and a low use of antimicrobials in general. Furthermore, it investigated whether the current consumption of aminopenicillins selects for ESC-producing E. coli and whether certain clones or plasmids spread from imported parent flocks to the meat. ESC-producing E. coli was isolated using MacConkey broth with 1 mg/L of ceftriaxone. ESC genes were identified using polymerase chain reaction and sequencing. Isolates with blaCMY-2 were subtyped by pulsed-field gel electrophoresis (PFGE), phylotyping, and antimicrobial susceptibility testing. Selected isolates were used as donors in filter-mating experiments, multilocus sequence typing (MLST), and plasmid replicons were typed. Aminopenicillin use at the farm (not flock) level was obtained from VetStat, a database for mandatory registration of veterinary prescriptions in Denmark. ESC-producing E. coli occurred in 93% (27/29) of broiler parent farms in 2011, 27% (53/197) of broiler flocks in 2010, and 3.3% (4/121) of Danish retail broiler meat in 2009 and 8.6% (16/187) in 2010. The ESC producing E. coli contained blaCMY-2, blaSHV-2 or blaCTX-M-1. Isolates with blaCMY-2 represented 35 PFGE groups. One group dominated (39 isolates) and included isolates with indistinguishable PFGE patterns from parents, broilers, and meat. Most blaCMY-2 isolates were susceptible to non-β-lactams, and blaCMY-2 was mostly present on horizontally transferable incI1 or incK plasmids. Phylogroup D was most common and E. coli MLST types previously found in humans were observed. Broiler farms with registered aminopenicillin use had significantly higher occurrence of ESC E. coli. ESC-producing E. coli from flocks of imported broiler parents spread clonally and horizontally to broiler meat (including potentially human pathogenic types) even in a country with no cephalosporin use. Use of aminopenicillins may influence the persistence of ESC-producing E. coli in the broiler production, but other factors should be investigated.