XbaI Pulsed-field Gel Electrophoresis Research Articles

KPC-Producing Enterobacterales from Douro River, Portugal-Persistent Environmental Contamination by Putative Healthcare Settings.

Carbapenemase-producing Enterobacterales (CPE) are a growing concern, representing a major public health threat to humans, especially in healthcare settings. In the present study, we evaluated the persistent contamination by carbapenem-resistant Enterobacterales in water from Douro River, Portugal. KPC-producing Enterobacterales were detected in five water samples separated chronologically by 15 days each. Susceptibility testing was performed by disk-diffusion-method according to Clinical and Laboratory Standards Institute (CLSI), phenotypic carbapenemase activity was evaluated by carbapenem inactivation method, presumptive identification of the isolates was performed by CHROMagar orientation and confirmed by API-20E. Carbapenemase genes were screened by PCR and the clonality of all isolates was assessed by XbaI-Pulsed Field Gel Electrophoresis (PFGE). Fifteen KPC-producing Enterobacterales isolates were selected, identified as multidrug-resistant and showed a resistance profile to non-beta-lactam antibiotics: sulfamethoxazole + trimethoprim (7/15), ciprofloxacin (3/15), fosfomycin (3/15) and chloramphenicol (2/15). Isolates were identified as (6) Escherichia coli and (9) Klebsiella pneumoniae. Our results suggest a punctual contamination with KPC-producing Enterobacterales continued through the time. The absence of clonality between the isolates suggests a circulation of mobile genetic element harbouring KPC gene in the origin of contamination. This work provides a better understanding on the impacts of water pollution resulting from human activities on aquatic environments.

ANTIMICROBIAL RESISTANCE AND MOLECULAR TYPING OF Salmonella spp. ISOLATED FROM POULTRY IN KHARTOUM STATE, SUDAN

Salmonella is considered one of the main foodborne pathogens. In this study a total of 38 Salmonella isolates were recovered from 679 (5.6 %) specimens collected within Khartoum State. The specimens included, intestinal contents 11.4% (14/160) egg 7% (7/99), liver 5% (5/100) skin 5% (10/200), feed 0% (0/40%), water 0% (0/100).. Serotyping revealed the presence of eight serovars: Kentucky 11 (28.9%), Stanleyville 8 (21.05%), Virchow 6 (16%), Alachua 5 (13.16%), Blockley 4 (10.53%), Hadar 2 (5.26%), Typhimurium 1 (2.63%) and Havana 1 (2.63%). Antibiotic resistance profile, using disc diffusion method, indicated that all isolates were sensitive to apramycin, chloramphenicol, cefoperazone and cefotaxime. The 38 isolates were found to be resistant to tetracycline (52.6%), nalidixic acid (50.0%), compound sulfonamide (44.7%), sulfamethoxazole-trimethoprim (31.6%), streptomycin (26.3%), gentamycin (15.8%), neomycin (15.8%), furazolidone (7.9%), ampicillin (5.3%), ciprofloxacin (5.3%), amikacin (2.6%), amoxicillin-clavulanic acid (2.6%), ceftazidime (2.6%), and colistin (2.6%) in decreasing order. Only 20 isolates (52.6%) demonstrated multiple drug resistance. Four of the Salmonella isolates were untypable by XbaI Pulsed Field Gel Electrophoresis (PFGE) technique however the 33 typed isolates were differentiated into 22 PFGE patterns. This study aimed to determine the prevalence, antimicrobial resistance and molecular typing of Salmonella spp. form poultry.

Plasmidic resistance to colistin mediated by mcr-1 gene in Escherichia coli clinical isolates in Argentina: A retrospective study, 2012-2018.

ABSTRACTObjective. To describe the resistance profile and the genetic characteristics of Escherichia coli isolates that harbor the mobilizable colistin resistance gene mcr-1 in Argentina.Methods. This was a retrospective study of 192 E. coli isolates positive for mcr-1 obtained from 69 hospitals of Buenos Aires City and 14 Argentinean provinces in 2012 – 2018. The antimicrobial susceptibility was performed by agar diffusion, broth macrodilution, and/or agar dilution. Standard polymerase chain reaction (PCR) was performed to detect resistance genes and incompatibility groups; specific PCR was applied to discriminate between blaCTX-M allelic groups and mcr-1.5 variant. The genetic relatedness among isolates was evaluated by XbaI-pulsed field gel electrophoresis and multilocus sequence typing in a subset of isolates.Results. All E. coli isolates showed minimal inhibitory concentrations to colistin ≥ 4μg/mL; nearly 50% were resistant to third-generation cephalosporins, with CTX-M-2 being the main extended-spectrum β-lactamase detected. Five E. coli were carbapenemase-producers (3 NDM, 2 KPC). The mcr-1.5 variant was detected in 13.5% of the isolates. No genetic relationship was observed among the mcr-1-positive E. coli clinical isolates, but a high proportion (164/192; 85.4%) of IncI2 plasmids was detected.Conclusions. The presence of IncI2 plasmids among highly diverse E. coli clones suggests that the mcr-1 gene’s wide distribution in Argentina may be driven by the horizontal transmission of IncI2 plasmids.

Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 in Broiler Chicken Supply Chain and Patients, China, 2010-2016.

Salmonella enterica serovar Kentucky (S. Kentucky) sequence type 198 has emerged as a global zoonotic pathogen. We explored Salmonella enterica serovar Kentucky ST198 samples from the broiler chicken supply chain and patients between 2010 and 2016. Here, we collected 180 S. Kentucky isolates from clinical cases and the poultry supply chain. We performed XbaI pulsed-field gel electrophoresis and multilocus sequence typing. We assessed mutations in the quinolone resistance-determining regions and screened for the presence of the Salmonella genomic island 1 (SGI1). We determined that 63 (35.0%) of the 180 isolates were S. Kentucky ST198. Chinese strains of S. Kentucky ST198 have a high transmission of ciprofloxacin resistance (38/63, 60.3%) and a high risk of multidrug resistance. The quinolone resistance of the S. Kentucky ST198 strain found in China may be due to mutations in its quinolone resistance-determining region. Our study firstly revealed that ciprofloxacin-resistant S. Kentucky ST198 strains can undergo cross-host transmission, thereby causing a serious foodborne public health problem in China.

535. Multicenter Study of the Prevalence of Rectal Colonization by Carbapenem-Resistant Enterobacteriaceae in Patients Admitted to the Intensive Care Units of 7 Major Hospitals in Kuwait

BackgroundThe emergence of carbapenem-resistant Enterobacteriaceae (CRE) has become an important epidemiological change in infectious diseases in the last 10 years. The gut is an important reservoir for these isolates thereby creating an opportunity for dissemination in a hospital setting especially the intensive care units (ICUs). The objective of this study was to investigate the colonization rates of patients, by CRE, admitted to the ICUs of 7 teaching hospitals.MethodsRectal swabs were collected during July 2017 to November 2018 from all patients on the day of ICU admission and 1 week after in each hospital. The samples were screened by direct plating on MacConkey agar containing 10-μg meropenem. Bacterial species identification was performed using the VITEK-2 system. The minimum inhibitory concentrations (MICs) of 14 antibiotics were determined by using Etest. Genes encoding carbapenem resistance was detected by PCR and sequencing. Their clonal relationship was determined by pulsed-field gel electrophoresis (PFGE).ResultsA total of 2580 Enterobacteriaceae were isolated from all patients. Seventy-four (2.9%) were confirmed as CRE most of which were from patients in Adan (AH: 36.5%) and Mubarak (MH: 46%) hospitals. Sixty (81.1%) harbored one or more of the tested carbapenemases genes. Forty-six (62.2%) carried blaOXA-181, 9 (12.2%) blaOXA-48, one blaKPC-2, while 14 (18.9%) carried 2 genes. Combinations of blaKPC-2 and blaOXA-181 genes were found in 5 (6.8%), blaVIM-1 and blaOXA-181 in 4 (5.4%), blaNDM-5 and blaOXA-181 in 3 (4.1%) and blaNDM-1 and blaOXA-181 in 2 (3.3%). The XbaI PFGE profile-based Dendrogram, at 85% similarity criterion, resolved 7 pulsotypes among isolates carrying blaOXA-181 in AH and MH designated A, B, C, D, E, F, and G. Further analysis revealed that 7 subpulsotypes A1, A2, A5, A6, C1, C2, and E1 were from unit D in the medical ICU of MH and A3, A4, B1, B3, D1, D2, D3, D4, F1, F2, F3, G1, and G2 were from surgical/medical ICUs in AH. 100% similarity was demonstrated among 8 isolates from AH and 2 from MH.ConclusionThe prevalence of rectal colonization by CRE in the ICU patients was lower than expected. Detection of blaOXA-181 variety and blaNDM-5 is new to the milieu of genes so far described in isolates from Kuwait.Disclosures All authors: No reported disclosures.

Salmonella Infantis in Broiler Flocks in Slovenia: The Prevalence of Multidrug Resistant Strains with High Genetic Homogeneity and Low Biofilm-Forming Ability.

For almost a decade, the number of Salmonella enterica subspecies enterica serovar Infantis-positive broiler flocks has been steadily increasing in Slovenia, doubling the number of positive holdings in only a few years. Since multidrug resistant S. Infantis isolates are highly prevalent in the broiler meat industry and may represent a public health concern through the food chain, we aimed to investigate the antimicrobial susceptibility, genetic diversity, and biofilm-forming ability of S. Infantis from Slovenian broiler flocks. A total of 87 S. Infantis strains isolated from broiler faeces in the period between 2007 and 2013 were studied. The samples originated from 41 farms which were subcontractors of three major food business operators and from two autonomously operating holdings (farms). Isolates were phenotypically tested for their susceptibility to 14 antimicrobials from nine classes by determining the minimum inhibitory concentration with the microdilution method. Only 8% of the isolates were susceptible to all of the antimicrobial agents tested, while 88.5% of the isolates were multidrug resistant, with the most common resistance pattern CipNxSSuT (65.5%) followed by CipNxSuT (17.2%). Pulsed-field gel electrophoresis (PFGE) divided the strains into five clusters (A-E) comprising 16 distinct XbaI PFGE profiles. Sixty-five out of 87 isolates were grouped in clusters A and B, with the predominant PFGE profiles A1 and B1 encompassing 33 and 28 isolates, respectively. A vast majority of the isolates (75/87) showed >90% PFGE profile similarity. The biofilm-forming capacity of the tested isolates, determined with crystal violet assay in polystyrene microwell plates, was generally weak. The average biofilm formation for persistent strains was higher than for presumably nonpersistent strains; however, the difference was not significant. It seems that S. Infantis persistence on broiler farms is more related to its widespread occurrence in the broiler production chain and ineffective disinfection protocols than to its ability to form biofilm.

OXA-48-Like-Producing Klebsiella pneumoniae in Southern Spain in 2014-2015.

The aim of this study was to characterize the population structure of 56 OXA-48-like-producing Klebsiella pneumoniae isolates, as well as extended-spectrum β-lactamase (ESBL) and carbapenemase genes, recovered in 2014 and 2015 from 16 hospitals in southern Spain. XbaI pulsed-field gel electrophoresis and multilocus sequence typing were performed to assess clonal relatedness. Representative isolates belonging to OXA-48-like-producing and CTX-M-15-coproducing pulsotypes were selected for characterization of blaOXA-48-like- and blaCTX-M-15-carrying plasmids by PCR-based replicon typing, IncF subtyping, whole-genome sequencing analysis, and typing of Tn1999 structures. Forty-three OXA-48-producing isolates (77%) were recovered from clinical samples and 13 from rectal swabs. All isolates showed ertapenem MIC values of ≥1 mg/liter, although 70% remained susceptible to imipenem and meropenem. Forty-nine isolates (88%) produced OXA-48, 5 produced OXA-245, and 2 produced OXA-181. Twenty-eight different pulsotypes (5 detected in more than 1 hospital) and 16 sequence types (STs) were found. The most prevalent clones were ST15 (29 isolates [52%]) and ST11 (7 isolates [13%]). Forty-five (80%) isolates were also blaCTX-M-15 carriers. The blaCTX-M-15 gene was mostly (82%) located on IncR plasmids, although ST15 and ST11 isolates also carried this gene on IncF plasmids. The composite transposon variant Tn1999.2-like was the most frequent. Among ST15 and ST11 isolates, different transposon variants were observed. The blaOXA-48 gene was mainly located on IncL plasmids, although IncM plasmids were also observed. The spread of OXA-48-like-producing K. pneumoniae in southern Spain is mainly due to ST15 and ST11 clones. Variation within clonal lineages could indicate different acquisition events for both ESBL and carbapenemase traits.

Erratum: Confirming TDP2 mutation in spinocerebellar ataxia autosomal recessive 23 (SCAR23).

[This corrects the article on p. e262 in vol. 4, PMID: 30109272.].

The characterization of Bordetella pertussis strains isolated in the Central-Western region of Brazil suggests the selection of a specific genetic profile during 2012–2014 outbreaks

Pertussis is a worldwide acute respiratory disease caused by the bacterium Bordetella pertussis. Despite high vaccine coverage, the bacterium continues to circulate in populations and is still one of the most common vaccine-preventable diseases. In Brazil, pertussis incidence has presented a significant decrease since 1990 but since 2011 a sudden increase in incidence has been observed. Thus, the aim of this study was to perform a molecular epidemiological characterization of B. pertussis strains isolated in the Central-Western region (specifically in Distrito Federal) of Brazil from August 2012 to August 2014. During this period, 92 B. pertussis strains were isolated from the outbreaks. All strains were characterized by serotyping and XbaI pulsed-field gel electrophoresis profiles. From August to December 2012, the most prevalent serotype observed was 1,3 (13/17). During 2013 the prevalence of serotype 1,3 decreased (13/30) and from January 2014 to August 2014 the most prevalent serotype was 1,2 (33/45). Fourteen PFGE profiles were identified. Of these, BP-XbaI0039 prevalence increased from 3/17 in 2012 to 10/30 in 2013, and 35/45 in 2014. These results evidence the selection of a specific genetic profile during this period, suggesting the occurrence of a bacterial genomic profile with high circulation potential.

Outbreak of Salmonella Oslo Infections Linked to Persian Cucumbers - United States, 2016.

In April 2016, PulseNet, the national molecular subtyping network for foodborne disease surveillance, detected a multistate cluster of Salmonella enterica serotype Oslo infections with an indistinguishable pulsed-field gel electrophoresis (PFGE) pattern (XbaI PFGE pattern OSLX01.0090).* This PFGE pattern was new in the database; no previous infections or outbreaks have been identified. CDC, state and local health and agriculture departments and laboratories, and the Food and Drug Administration (FDA) conducted epidemiologic, traceback, and laboratory investigations to identify the source of this outbreak. A total of 14 patients in eight states were identified, with illness onsets occurring during March 21-April 9, 2016. Whole genome sequencing, a highly discriminating subtyping method, was used to further characterize PFGE pattern OSLX01.0090 isolates. Epidemiologic evidence indicates Persian cucumbers as the source of Salmonella Oslo infections in this outbreak. This is the fourth identified multistate outbreak of salmonellosis associated with cucumbers since 2013. Further research is needed to understand the mechanism and factors that contribute to contamination of cucumbers during growth, harvesting, and processing to prevent future outbreaks.

Influence of Pigskin on Salmonella Contamination of Pig Carcasses and Cutting Lines in an Italian Slaughterhouse.

Ninety pig carcasses and twenty one food contact surfaces (FCSs) were tested for Salmonella in a slaughterhouse processing ca. 380 pigs/h between 2014-2015. Sampling was performed during seven sessions. Four carcass sites of 100 cm2 each (back, belly, jowl externally, and the diaphragmatic area internally) were swabbed after evisceration. Meat conveyors and dressing tables were tested swabbing areas of 200 to 400 cm2. After pre-enrichment in buffered peptone water, samples were tested by Salmonella MDS® assay and the presumptive positives were confirmed by the ISO 6579 method. Salmonella isolates were serotyped following the Kauffman-White-Le Minor scheme and genotyped by XbaI pulsed field gel electrophoresis. Salmonella was isolated from 16/90 [17.8%; confidence interval (CI) 95%=11.2-26.9] carcasses and 4/21 (19.0%; CI 95%=7.7-40.0) FCSs. Four serovars were identified on carcasses. S. enterica 4,[5],12:i:-was the most prevalent (43.75%), followed by S. Rissen (31.25%), S. Derby (12.5%) and S. Bovismorbificans (12.5%). Two serovars were found on FCSs, namely S. Derby (75%) and S. Livingstone (25%). During one sampling session, a failure in carcass dehairing occurred and caused significantly higher prevalence of carcass contamination (60%) than in the remaining sessions. Moreover, in the same session, Salmonella prevalence was marginally significantly higher on FCSs than in the remaining sampling days, suggesting that dehairing affects contamination not only on carcasses, but also on the working surfaces.

Genetic & virulence profiling of ESBL-positive E. coli from nosocomial & veterinary sources

CTX-M genes are the most prevalent ESBL globally, infiltrating nosocomial, community and environmental settings. Wild and domesticated animals may act as effective vectors for the dissemination of CTX-producing Enterobacteriaceae. This study aimed to contextualise blaCTX-M-14-positive, cephalosporin-resistant Enterobacteriaceae human infections and compared resistance and pathogenicity markers with veterinary isolates.Epidemiologically related human (n=18) and veterinary (n=4) blaCTX-M-14-positive E. coli were fully characterised. All were typed by XbaI pulsed field gel electrophoresis and ST. Chromosomal/plasmidic locations of blaCTX-M-14 were deduced by S1-nuclease digestion, and association with ISEcp1 was investigated by sequencing. Conjugation experiments assessed transmissibility of plasmids carrying blaCTX-M-14. Presence of virulence determinants was screened by PCR assay and pathogenicity potential was determined by in vitro Galleria mellonella infection models.84% of clinical E. coli originated from community patients. blaCTX-M-14 was found ubiquitously downstream of ISEcp1 upon conjugative plasmids (25–150kb). blaCTX-M-14 was also found upon the chromosome of eight E. coli isolates. CTX-M-14-producing E. coli were found at multiple hospital sites. Clonal commonality between patient, hospitals and livestock microbial populations was found. In vivo model survival rates from clinical isolates (30%) and veterinary isolates (0%) were significantly different (p<0.05). Co-transfer of blaCTX-M-14 and virulence determinants was demonstrated.There is evidence of clonal spread of blaCTX-M-14-positive E. coli involving community patients and farm livestock. blaCTX-M-14 positive human clinical isolates carry a lower intrinsic pathogenic potential than veterinary E. coli highlighting the need for greater veterinary practices in preventing dissemination of MDR E. coli among livestock.

Antimicrobial Drug Resistance and Molecular Typing of Salmonella enterica Serovar Rissen from Different Sources.

Salmonella enterica serovar Rissen is one of the most common serovars found in pigs and pork products in different countries, including Spain. However, information on the molecular bases of antimicrobial drug resistance and the population structure of Salmonella Rissen from different sources in Spain is limited. The present study focused on 84 isolates collected in Spain from pig and beef carcasses, foods and clinical samples associated with sporadic cases of gastroenteritis, and one outbreak. The majority of the isolates were resistant to tetracycline (73.8%), mainly conferred by tet(A). Resistances to streptomycin (aadA1-like, aadA2, and strAB), sulfonamides (sul1, sul2, and sul3), trimethoprim (dfrA1-like and dfrA12), ampicillin (blaTEM-1-like), and chloramphenicol (cmlA1-like) were also detected, with frequencies ranging from 12% to 20.2%. Most of the identified genes were carried by integrons, including three class 1 integrons of the sul1 type, a class 1 integron of the sul3 type, and the class 2 integron of Tn7. Two sul1 integrons, the sul3 integron, and the class 2 integron are first reported in Salmonella Rissen. Typing of the isolates with XbaI pulsed-field gel electrophoresis detected a major clone, which was circulating in humans and animals during the past decade, and was responsible for the outbreak. The obtained results are relevant for food safety and public health.

Salmonella surveillance on fresh produce in retail in Turkey

Although Turkey is one of the major producers of fruits and vegetables in the world, there has been no information available on the prevalence of pathogens in fresh produce. To fill this gap, we collected 503 fresh produce samples including tomato, parsley, iceberg lettuce, green-leaf lettuce and five different fresh pepper varieties (i.e., green, kapya, bell, mazamort and Charleston) from 3 major districts within 9 supermarkets and 3 bazaars in Ankara, Turkey to investigate the presence of Salmonella. Salmonella was detected in 0.8% (4/503) of samples by conventional culturing method with molecular confirmation conducted through polymerase chain reaction (PCR). For further characterization of isolates, serotyping, antimicrobial susceptibility testing, multi-locus sequence typing (MLST; aroC, thrA, purE, sucA, hisD, hemD and dnaN) and pulsed-field gel electrophoresis (PFGE) were performed. Salmonella enterica subsp. enterica serotypes Anatum, Charity, Enteritidis and Mikawasima were isolated from two parsley, one pepper and one lettuce samples, respectively. MLST resulted in 4 sequence types (STs) for each serotype, including one novel ST for serotype Mikawasima. Similarly, PFGE revealed four different XbaI PFGE patterns. The results of this survey, obtained by the most common subtyping methods (i.e. serotyping, MLST and PFGE) worldwide, contributes to the development of a national database in Turkey, which is essential for investigating the evolutionary pathways, geographical distribution and genetic diversity of Salmonella strains.

Molecular Typing of Salmonella Isolates in Poultry by Pulsed-Field Gel Electrophoresis in Iran

Background: Salmonella is one of the most widespread zoonotic enter pathogenic microorganisms found in the global food chain. Poultryand Poultry products have been identified as one of the important foodborne sources of Salmonella. Pulsed-Field Gel Electrophoresis (PFGE) is a gold standard typing method for identification of Salmonella isolates during outbreaks and epidemiological investigations. Objectives: The aim of this study was to carry out molecular typing of Salmonella enterica spp. by PFGE technique. Materials and Methods: All 47 Salmonella isolates were serotyped and then subjected to PFGE. Total isolates were analyzed by means of the molecular technique XbaI PFGE. Results: In the current work, PFGE and serotyping were used to subtype 47 Salmonella isolates belonging to 22 different serotypes and derived from poultry. Thirty-nine PFGE patterns out of 47 isolates were obtained. The Discrimination Index (DI) by serotyping (0.93) was lower than PFGE (DI = 0.99). Conclusions: In conclusion, molecular methods such as PFGE can be used for epidemiological characterization of Salmonella serotypes.

Prevalence and characterization of Salmonella species in entire steps of a single integrated broiler supply chain in Korea

The purpose of this study was to investigate the distribution of Salmonella species in an integrated broiler supply chain in Korea. A total of 1,214 samples from various steps of an integrated broiler production company including broiler breeder farms, broiler farms, broiler trucks, slaughterhouse, and retail chicken meats were collected and investigated. Salmonella was detected in 195 of the samples. The highest prevalence of Salmonella was observed in broiler transporting trucks (71.43%), followed by the slaughterhouse (63.89%) and broiler farms (16.05%). Salmonella Hadar was the most frequently isolated serotype (83.08%). All Salmonella Hadar isolates investigated in this study with pulsed-field gel electrophoresis showed the same XbaI pulsed-field gel electrophoresis pulsotype.

Distribution ofEscherichia colistrains harbouring Shiga toxin-producingE. coli(STEC)-associated virulence factors (stx1, stx2, eae, ehxA) from very young calves in the North Island of New Zealand

The objective of this study was to determine the distribution of Shiga toxin-producing Escherichia coli (STEC) virulence markers (stx1, stx2, eae, ehxA) in E. coli strains isolated from young calves aged fewer than 7 days (bobby calves). In total, 299 recto-anal mucosal swabs were collected from animals at two slaughter plants and inoculated onto tryptone bile X-glucuronide and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Isolates were analysed using multiplex polymerase chain reaction to detect stx1, stx2, eae and ehxA genes. The most common combination of virulence markers were eae, ehxA (n = 35) followed by eae (n = 9). In total, STEC and atypical enteropathogenic E. coli (aEPEC) were isolated from 8/299 (2·6%) and 37/299 (12·3%) calves, respectively. All the isolates could be assigned to 15 genotype clusters with >70% similarity cut-off using XbaI pulsed-field gel electrophoresis. It may be concluded that healthy calves from the dairy industry are asymptomatic carriers of a diverse population of STEC and aEPEC in New Zealand.

Characterization ofSalmonellaspp. Clinical Isolates in Gyeongsangbuk-do Province, 2012 to 2013

Background: Extended-spectrum cephalosporins and fluoroquinolones are important antimicrobials for treating invasive salmonellosis, and emerging resistance to these antimicrobials is of paramount concern. Methods: A total of 30 Salmonella spp. clinical isolates recovered in Gyeongsangbuk-do from 2012 to 2013 were characterized using antibiotic resistance profiles and pulsed-field gel electrophoresis (PFGE). Results: A high prevalence of multidrug-resistant isolates, mainly showing an ampicillin, nalidixic acid, chloramphenicol resistance pattern, was observed. Four extended-spectrum β-lactamase (ESBL)-producing isolates (3 CTX-M-15 isolates and 1 CTX-M-27 isolate) were found. The blaCTX-M-27 gene was carried by an IncF conjugative plasmid in the S. Infantis isolate. The blaCTX-M-15 gene were carried by an IncF (2 isolates) or IncHI2 (1 isolate) conjugative plasmid in S. Enteritidis. In addition, a single mutation of GyrA, Ser83Thr (1 isolates), Asp87Tyr (9 isolates), Asp87Gly (4 isolates), and Asp87Leu (3 isolates), was detected in nalidixic acid-resistant Salmonella spp. isolates. XbaI PFGE analysis of all isolates revealed more than 19 different pulsotypes. The most common S. Enteritidis PFGE pattern (SEGX01.003) was associated with a larger number of cases of invasive salmonellosis than all other patterns. Conclusion: The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize disease prevention. (Ann Clin Microbiol 2014;17:50-57)

Optimization of Pulse-Field Gel Electrophoresis for Subtyping of Klebsiella pneumoniae

A total of 110 strains of Klebsiella pneumoniae were used to optimize pulsed-field gel electrophoresis (PFGE) for subtyping of K. pneumoniae. For optimization of electrophoresis parameters (EPs) of XbaI-PFGE, 11 isolates were analyzed with XbaI digestion using three EPs. The EP of a switch time of 6 to 36 s for 18.5 h gave clearest patterns and was declared the optimal EP for XbaI PFGE of K. pneumoniae. By software analysis and pilot study, AvrII was chosen as another PFGE enzyme. Both XbaI- and AvrII-PFGE gave D-values higher than 0.99 for 69 K. pneumoniae isolated from different sources. Our results also showed good typeability, reproducibility of both XbaI- and AvrII-PFGE for K. pneumoniae subtyping. Furthermore, the established PFGE method also had good discriminatory power to distinguish outbreak K. pneumoniae strains and a high degree of consistency with multilocus sequence typing method. A rapid PFGE protocol was established here, which could be used for genotyping and other researches of K. pneumoniae.

Salmonella entericaserovarTyphimurium Virulence-Resistance Plasmids Derived from the pSLT Carrying Nonconventional Class 1 Integrons withdfrA12Gene in Their Variable Region andsul3in the 3′ Conserved Segment

Drug-resistant derivatives of serovar-specific virulence plasmids, such as pSLT, in clinically-relevant Salmonella enterica serovar Typhimurium strains, represent a threat for human health. We have analysed 14 S. Typhimurium isolates recovered in Italy and the United Kingdom from swine and from cases of human infection for the presence of virulence-resistance (VR) plasmids. They were negative for the multidrug resistance (MDR) region of the Salmonella genomic island 1 (SGI1), but expressed resistance to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfamethoxazole, and tetracyclines. The isolates were characterised by XbaI pulsed-field gel electrophoresis, multilocus sequence typing, and detection of resistance and virulence determinants (PCR/sequencing). Identification of VR plasmids was accomplished by PCR detection of bla genes (encoding ampicillin resistance), class 1 integrons and the pSLT virulence gene spvC. Plasmid analyses were performed by alkaline lysis, S1-nuclease digestion, replicon typing, conjugation, restriction analyses, and Southern blot/hybridization. Two blaOXA-1 positive isolates contained pSLT-derived plasmids related to pUO-StVR2. In nine isolates, eight from swine and one from a patient, MDR-conferring-IncFII-VR plasmids were detected. They contained the blaTEM-1 gene as well as a nonconventional class 1 integron with dfrA12-aadA2 gene cassettes in its variable region, and a sul3 gene in the 3' conserved segment. Restriction analysis suggested a novel pSLT variant. The results obtained underline the role of swine as a potential reservoir for the blaTEM-1-IncFII-plasmids. The occurrence and spread of virulence- and MDR-conferring plasmids should be considered as a potential public health problem.