Similar Pulsed-field Gel Electrophoresis Research Articles

Whole-Genome Sequencing of Shiga Toxin-Producing Escherichia coli for Characterization and Outbreak Investigation.

Shiga toxin-producing Escherichia coli (STEC) causes high frequencies of foodborne infections worldwide and has been linked to numerous outbreaks each year. Pulsed-field gel electrophoresis (PFGE) has been the gold standard for surveillance until the recent transition to whole-genome sequencing (WGS). To further understand the genetic diversity and relatedness of outbreak isolates, a retrospective analysis of 510 clinical STEC isolates was conducted. Among the 34 STEC serogroups represented, most (59.6%) belonged to the predominant six non-O157 serogroups. Core genome single nucleotide polymorphism (SNP) analysis differentiated clusters of isolates with similar PFGE patterns and multilocus sequence types (STs). One serogroup O26 outbreak strain and another non-typeable (NT) strain, for instance, were identical by PFGE and clustered together by MLST; however, both were distantly related in the SNP analysis. In contrast, six outbreak-associated serogroup O5 strains clustered with five ST-175 serogroup O5 isolates, which were not part of the same outbreak as determined by PFGE. The use of high-quality SNP analyses enhanced the discrimination of these O5 outbreak strains into a single cluster. In all, this study demonstrates how public health laboratories can more rapidly use WGS and phylogenetics to identify related strains during outbreak investigations while simultaneously uncovering important genetic attributes that can inform treatment practices.

Characterization of small plasmids carrying florfenicol resistance gene floR in Actinobacillus pleuropneumoniae and Pasteurella multocida isolates from swine in China.

Actinobacillus pleuropneumoniae and Pasteurella multocida are two important bacterial pathogens in swine industry. In the present study, resistance profiles of nine commonly used antibiotics of A. pleuropneumoniae and P. multocida isolates of swine origin from different regions of China were investigated by determination of minimum inhibitory concentrations (MICs). In addition, genetic relationship of the florfenicol-resistant A. pleuropneumoniae and P. multocida isolates was determined by pulsed-field gel electrophoresis (PFGE). The genetic basis of florfenicol resistance in these isolates were explored by floR detection and whole genome sequencing. High resistance rates (>25%) of florfenicol, tetracycline and trimethoprim- sulfamethoxazole were observed for both bacteria. No ceftiofur- and tiamulin- resistant isolates were detected. Furthermore, all the 17 florfenicol-resistant isolates (nine for A. pleuropneumoniae and eight for P. multocida) were positive for floR gene. The presence of similar PFGE types in these isolates suggested that clonal expansion of some floR-producing strains occurred in the pig farms from same regions. WGS and PCR screening showed that three plasmids, named pFA11, pMAF5, and pMAF6, were the cargos of the floR genes in the 17 isolates. Plasmid pFA11 exhibited novel structure and carried several resistance genes, including floR, sul2, aacC2d, strA, strB, and bla ROB - 1. Plasmids pMAF5 and pMAF6 were presented in A. pleuropneumoniae and P. multocida isolates from different regions, suggesting horizontal transfer of the two plasmids are important for the floR dissemination in these Pasteurellaceae pathogens. Further studies of florfenicol resistance and its transfer vectors in Pasteurellaceae bacteria of veterinary origin are warranted.

Carbapenem-resistant Klebsiella pneumoniae: Risk Factors for Isolation Among Hospitalized Patients

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important healthcare-associated pathogen. This study aimed to identify factors associated with CRKP isolation among hospitalized patients, describe molecular epidemiology, and mortality associated with CRKP isolation. Methods: We performed a case-control study at two university-affiliated teaching hospitals. We included 150 patients (30 cases and 120 controls). Each patient with CRKP, a case-patient, was matched with four controls by admission facility, admission date, age, and sex. Controls, patients without CRKP, were randomly selected from a computerized list of inpatients whose admission date was the same as that of the case, within 48 hours of the date of the initial positive culture. We calculated the risk of in-hospital death as the number of deaths divided by the number of cases and evaluated the risk of mortality associated with the site of positive culture. Molecular epidemiology investigation using comparison of restricted DNA patterns of CRKP by pulsed-field gel electrophoresis (PFGE) was conducted. Results: A greater proportion of cases than controls had undergone an invasive procedure, including use of a central vein catheter, or mechanical nutrition by tube feeding. Pre-admission treatment within two months with the following antibiotic classes was associated with CRKP isolation: carbapenems, fluoroquinolones, anti-pseudomonal penicillins, and cephalosporins. The molecular analysis indicated that over 90% of isolates shared similar PFGE patterns. CRKP isolation was associated with significantly higher in-hospital mortality in comparison to controls. Positive cultures from sites other than urine were associated with substantially higher mortality than was a positive urine culture (RR= 4.0). Conclusions: The use of multiple broad-spectrum antibiotics, multiple comorbid conditions and poor performance status are important risk factors for developing CRKP in a hospitalized population.

Multistate reptile- and amphibian-associated salmonellosis outbreaks in humans, United States, 2009-2018.

Non-typhoidal Salmonella cause an estimated 1.4 million human illnesses, 26,000 hospitalizations and 400 deaths annually in the United States. Approximately 11% of these infections are attributed to animal contact. Reptiles and amphibians are known sources of salmonellosis; young children (aged <5 years) are disproportionately affected by reptile- and amphibian-associated salmonellosis (RAAS) outbreaks. We describe multistate RAAS outbreaks to characterize illnesses and inform prevention efforts. RAAS outbreaks were defined as ≥2 culture-confirmed human Salmonella infections with similar pulsed-field gel electrophoresis patterns and epidemiologic, laboratory or traceback evidence linking them to a common reptile/amphibian exposure. Data sources included the Animal Contact Outbreak Surveillance System; CDC Outbreak Response and Prevention Branch's outbreak management database; PulseNet, the national molecular subtyping network for foodborne disease surveillance in the United States; and the National Antimicrobial Resistance Monitoring System. Twenty-six RAAS outbreaks were reported during 2009-2018, resulting in 1465 illnesses and 306 hospitalizations. The outbreaks were associated with turtles (19), lizards (5), snakes (1) and frogs (1). Sixteen (61.5%) outbreaks were linked to small turtles (<4 inches), resulting in 914 illnesses. Forty-nine percent of outbreak-associated patients were aged <5 years. Of 362 patients/caregivers interviewed, 111 (30.7%) were aware that reptiles/amphibians can carry Salmonella. Among 267 patient isolates with antimicrobial susceptibility information, 20 (7.5%) were non-susceptible to ≥1 antibiotic used to treat human salmonellosis. RAAS outbreaks result in considerable morbidity, particularly among young children. Illnesses linked to small turtles are preventable through education, targeted outreach to caregivers and paediatricians, and when appropriate, enforcement. Historically, individual states and jurisdictions have enforced existing or promulgated new authorities to address outbreaks. Preventing future RAAS outbreaks requires addressing challenges related to the illegal sale/distribution of small turtles; and for legal reptile sales, providing information on RAAS risk to consumers at point of sale to support informed pet ownership decisions.

Molecular characterization of NDM-1-producing carbapenem-resistant E. cloacae complex from a tertiary hospital in Chongqing, China.

BackgroundThe aim of this study was to clarify the molecular characterization of NDM-1-producing carbapenem-resistant Enterobacter cloacae complex (CREL) at a teaching hospital in Chongqing, China.MethodsAntimicrobial susceptibility and resistance genes were analyzed. Epidemiological relationship was analyzed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Conjugation experiments were performed to determine the transferability of plasmids. Whole-genome sequencing (WGS) of strains was implemented, and the genetic environment of the bla NDM-1- and mcr-9-carrying plasmids was analyzed.ResultsA total of 10 bla NDM-1-positive CREL isolates were identified. All isolates harbored multiple resistance genes. ECL68 and ECL78 co-produce bla NDM-1 and mcr-9. Among the four different sequence types (STs) detected, ST1466 was assigned as a novel ST. Six isolates exhibited highly similar PFGE patterns. Conjugation assay proved that all plasmids containing bla NDM-1 or mcr-9 could be transferred to the recipient Escherichia coli. WGS indicated that bla NDM-1 genes were carried by diverse plasmids, including IncHI2/IncN, IncX3, and one unclassified plasmid type. The backbone structure of these plasmids is involved in replication initiation (repAB), partitioning (parABM), and conjugation/type IV secretion (tra/virB). Analysis of the genetic environment showed that bla NDM-1 in three plasmids exhibited a highly similar structure to protype Tn125. Co-existence of bla NDM-1 and the colistin resistance gene mcr-9 was detected in the two isolates, ECL68 and ECL78. In ECL68, bla NDM-1 and mcr-9 were present on the same plasmid while located in two separate plasmids in ECL78. The genetic environment of mcr-9 was organized as IS26-wbuC-mcr-9-IS903-pcoS-pcoE-rcnA-rcnR, and the two-component system encoding genes qseC and qseB was not found in two plasmids, which could explain mcr-9-harboring strains’ colistin susceptibility.ConclusionsWe first report a nosocomial outbreak of NDM-1-producing E. cloacae complex ST177 in China. Conjugative plasmids contributed to the horizontal transfer of antibiotic resistance genes. The prevalence and even coexistence of bla NDM-1 and mcr-9 may further threaten public health. Our results highlight further surveillance for bla NDM-1, and mcr-9 is essential to prevent its dissemination.

Clinical and Genomic Investigation of an International Ceftriaxone- and Azithromycin-Resistant Shigella sonnei Cluster among Men Who Have Sex with Men, Montréal, Canada 2017-2019.

ABSTRACTMultidrug-resistant (MDR) Shigella sonnei have become prevalent among men who have sex with men and have become a global public health concern. From June 2017 to April 2019, 32 men were infected with MDR S. sonnei acquired locally, in Montréal, which was suggestive of an outbreak. Antimicrobial susceptibility testing, whole-genome sequencing (WGS), phylogenetic analysis, antimicrobial resistance and virulence characterization, and association to international clusters were performed. The outbreak strain was ceftriaxone- and azithromycin-resistant due to the acquisition of blaCTX-M-27, and mphA and ermB genes, respectively, with reduced susceptibility to ciprofloxacin due to a single point mutation (gyrA S83L). One out of 27 patients treated with a fluoroquinolone experienced microbiological failure. Epidemiological evidence first supported by a rare unique MDR Shigella sonnei documented only in men in 2017 followed by similar pulsed-field gel electrophoresis profiles was confirmed by WGS. A core genome high-quality single-nucleotide variant (hqSNV)-based phylogeny found a median of 6 hqSNV differences among isolates. Virulence gene content was investigated, but no Shiga toxins were detected. An international cluster of highly related isolates was identified (PDS000019750.208) and belonged to the 3.7.29.1.4.1 S. sonnei genotype (Global III VN2.KH1.Aus). Genomic analysis revealed that this Montréal cluster was connected to other documented outbreaks in Australia, the United States, and the United Kingdom. This study highlights the urgent need for public health measures to focus on the prevention and the early detection of S. sonnei, since global transmission patterns of MDR strains is concerning and few antimicrobial treatment options are available.IMPORTANCE Shigella sonnei, an important foodborne pathogen, recently became a frequent sexually transmitted agent involved in large and persistent outbreaks globally among men who have sex with men. Most strains also harbor several multidrug-resistant (MDR) determinants of particular concern. This study characterizes an outbreak strain at the source of an important MDR cluster identified in Montréal in 2017. Associations were made to many high-profile international outbreaks, and the causative S. sonnei lineage of these clusters was identified, which was not evident in past reports. The worldwide occurrence of this strain is of concern since treatment with antimicrobials like ceftriaxone and azithromycin may not be effective, and rare microbiological failures have been documented in patients treated with ciprofloxacin. Our investigation highlights the threats of Shigella spp. infection and the necessity for antimicrobial susceptibility monitoring in order to mitigate S. sonnei’s impact on public health and to avoid transmission to other at-risk communities.

Identification of the Multiresistance Gene poxtA in Oxazolidinone-Susceptible Staphylococcus haemolyticus and Staphylococcus saprophyticus of Pig and Feed Origins.

Previous studies on the prevalence and transmission mechanism of oxazolidinone resistance gene poxtA in CoNS are lacking, which this study addresses. By screening 763 CoNS isolates from different sources of several livestock farms in Guangdong, China, 2018–2020, we identified that the poxtA was present in seven CoNS isolates of pig and feed origins. Species identification and multilocus sequence typing (MLST) confirmed that seven poxtA-positive CoNS isolates were composed of five ST64-Staphylococcus haemolyticus and two Staphylococcus saprophyticus isolates. All poxtA-positive Staphylococcus haemolyticus isolates shared similar pulsed-field gel electrophoresis (PFGE) patterns. Transformation assays demonstrated all poxtA-positive isolates were able to transfer poxtA gene to Staphylococcus aureus RN4220. S1-PFGE and whole-genome sequencing (WGS) revealed the presence of poxtA-carrying plasmids in size around 54.7 kb. The plasmid pY80 was 55,758 bp in size and harbored the heavy metal resistance gene czcD and antimicrobial resistance genes, poxtA, aadD, fexB and tet(L). The regions (IS1216E-poxtA-IS1216E) in plasmid pY80 were identified in Staphylococcus spp. and Enterococcus spp. with different genetic and source backgrounds. In conclusion, this was the first report about the poxtA gene in Staphylococcus haemolyticus and Staphylococcus saprophyticus, and IS1216 may play an important role in the dissemination of poxtA among different Gram-positive bacteria.

Emergence of mcr-3 carrying Escherichia coli in Diseased Pigs in South Korea

We examined the prevalence and molecular characteristics of mcr-3 carrying colistin-resistant Escherichia coli among cattle, pig, and chicken isolates in South Korea. Among a total of 185 colistin-resistant E. coli isolates determined in this study (47 from cattle, 90 from pigs, and 48 from chicken), PCR amplification detected mcr-3 genes in 17 isolates predominantly from diseased pigs. The mcr-3 genes were characterized as mcr-3.1 in 15 isolates and mcr-3.5 in 2 isolates. The mcr-3 gene was transferred to the E. coli J53 recipient strain from more than 50% of the mcr-3-carrying isolates. The mcr-3.1 and mcr-3.5 genes were identified predominantly in IncHI2 and IncP plasmids, respectively. Multi-locus sequence typing analysis revealed eight previously reported sequence types (ST), including ST1, ST10, and ST42. We identified isolates with similar pulsed-field gel electrophoresis patterns from diseased pigs in three farms. Besides, the isolates carried various virulence factors and demonstrated resistance to multiple antimicrobials, including β-lactams and quinolones. Further, the mcr-3.5 encodes three amino acid substitutions compared with mcr-3.1. To the best of our knowledge, this is the first report of pathogenic E. coli carrying mcr-3.5 in South Korea, which implies that mcr-3 variants may have already been widely spread in the pig industry.

Fourth Generation Cephalosporin Resistance Among Salmonella enterica Serovar Enteritidis Isolates in Shanghai, China Conferred by blaCTX–M–55 Harboring Plasmids

In this study, we investigated the pattern of antimicrobial resistance in Salmonella enterica serotype Enteritidis isolates in Shanghai, China from 2005 to 2014. We found the first isolates with resistance to the fourth-generation cephalosporin cefepime starting in 2010. Furthermore, we analyzed the epidemic characteristics and mechanisms of underlying cefepime resistance in S. Enteritidis isolates found from 2010. In total, 38 of 2,914 (1.30%) isolates were identified as cefepime-resistant S. Enteritidis (CRSE) isolates by Kirby–Bauer disk diffusion. Two isolates were from animal derived food sources; 36 isolates were from fecal samples of human patients with salmonellosis. Antimicrobial susceptibility testing using the agar dilution method revealed that all CRSE isolates showed additional resistances at least to ceftazidime, cefotaxime, and ampicillin. Additionally, pulsed-field gel electrophoresis (PFGE) profiles indicated that 89.47% of CRSE isolates also displayed similar PFGE patterns. Five types of β-lactamase genes, blaCTX–M (100.00%, 38/38), blaSHV (65.79%, 25/38), blaTEM (52.63%, 20/38), blaACC (18.42%, 7/38), and blaPSE (5.26%, 2/38) were detected by PCR and sequencing. Among blaCTX–M genes, blaCTX–M–55 was the dominant type (84.21%, 32/38). Conjugation and transformation experiments along with plasmid replicon typing revealed that blaCTX–M–55 was located on plasmids of various replicon types with sizes ranging from 76.8 to 138.9 kb. Plasmid sequence analysis also showed that the blaCTX–M–55 gene was mobilized mainly by the ISEcp1-blaCTX–M–55-ORF477 transposition unit and had its own ISEcp1-based promoter, which accelerated the expression and transmission of blaCTX–M–55. Analysis of whole genome sequences (Illumina) of one selected transformant SH12G706-C showed high similarity of the blaCTX–M–55 carrying plasmid with the IncI1 plasmid backbone p628-CTX-M of Klebsiella pneumoniae detected in 2010 in China. The present study demonstrated that the blaCTX–M–55 gene mobilized by ISEcp1- blaCTX–M–55-ORF477 was the main feature shared by CRSE isolates and seems to play an important role for transmission of cefepime resistance. The number of CRSE isolates is rising annually, and the strong dissemination ability of ISEcp1-blaCTX–M–55-ORF477-harboring plasmids among different species represents an important threat to the therapeutic effectiveness of cefepime.

Biofilm Production Ability, Virulence and Antimicrobial Resistance Genes in Staphylococcus aureus from Various Veterinary Hospitals.

Staphylococcus aureus (S. aureus) is one of the most clinically important zoonotic pathogens, but an understanding of the prevalence, biofilm formulation ability, virulence, and antimicrobial resistance genes of S. aureus from veterinary hospitals is lacking. By characterizing S. aureus in different origins of veterinary hospitals in Guangzhou, China, in 2019, we identified with the presence of S. aureus in pets (17.1%), veterinarians (31.7%), airborne dust (19.1%), environmental surfaces (4.3%), and medical device surfaces (10.8%). Multilocus sequence typing (MLST) and Staphylococcus protein A (spa) typing analyses demonstrated methicillin-sensitive S. aureus (MSSA) ST398-t571, MSSA ST188-t189, and methicillin-resistant S. aureus (MRSA) ST59-t437 were the most prevalent lineage. S. aureus with similar pulsed-field gel electrophoresis (PFGE) types distributed widely in different kinds of samples. The crystal violet straining assays revealed 100% (3/3) of MRSA ST59 and 81.8% (9/11) of MSSA ST188 showed strong biofilm formulation ability, whereas other STs (ST1, ST5, ST7, ST15, ST88, ST398, ST3154 and ST5353) showed weak biofilm production ability. Polymerase chain reaction (PCR) confirmed the most prevalent leucocidin, staphylococcal enterotoxins, ica operon, and adhesion genes were lukD-lukE (49.0%), sec-sel (15.7%), icaA-icaB-icaC-icaR (100.0%), and fnbB-cidA-fib-ebps-eno (100.0%), respectively. Our study showed that the isolates with strong biofilm production ability had a higher prevalence in clfA, clfB, fnbA and sdrC genes compared to the isolates with weak biofilm production ability. Furthermore, 2 ST1-MRSA isolates with tst gene and 1 ST88-MSSA isolate with lukS/F-PV gene were detected. In conclusion, the clonal dissemination of S. aureus of different origins in veterinary hospitals may have occurred; the biofilm production capacity of S. aureus is strongly correlated with ST types; some adhesion genes such as clfA, clfB, fnbA, and sdrC may pose an influence on biofilm production ability and the emergence of lukS/F-PV and tst genes in S. aureus from veterinary hospitals should raise our vigilance.

Prevalence, Antimicrobial Resistance, Virulence Genes and Genetic Diversity of Salmonella Isolated from Retail Duck Meat in Southern China.

Salmonella is an important cause of foodborne diseases. This study was undertaken to investigate the prevalence, serotype distribution, antimicrobial resistance, virulence genes, and genetic diversity of Salmonella isolates recovered from fresh duck meat obtained from retail markets in Southern China. In total, 365 samples of fresh duck meat were collected from retail markets in six different cities of Guangdong Province between May 2017 and April 2019. High levels of Salmonella contamination were detected in duck meat (151/365, 41.4%). Twenty-six different Salmonella serotypes were identified: S. Corvallis (n = 25, 16.6%), S. Kentucky (n = 22, 14.6%) and S. Agona (n = 20, 13.3%) were the most prevalent serotypes. All isolates were resistant to at least one antibiotic and 133 (88.1%) isolates exhibited multidrug resistance (MDR). Most (86.1%) Salmonella isolates carried seven classes of virulence-associated genes. This study showed the diversity of Salmonella serotypes and genotypes and the high prevalence of MDR isolates carrying multiple virulence-associated genes among isolates from duck meat obtained from retail markets in Southern China. Isolates from different districts had similar pulsed-field gel electrophoresis (PFGE) patterns indicating that circulating foodborne Salmonella constitutes a potential public health issue across different districts.

A prolonged multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales due to horizontal transmission of the IncN plasmid

A multispecies outbreak of IMP-6 carbapenemase-producing Enterobacterales (IMP-6-CPE) occurred at an acute care hospital in Japan. This study was conducted to understand the mechanisms of IMP-6-CPE transmission by pulsed-field gel electrophoresis (PFGE), multilocus sequence typing and whole-genome sequencing (WGS), and identify risk factors for IMP-6-CPE acquisition in patients who underwent abdominal surgery. Between July 2013 and March 2014, 22 hospitalized patients infected or colonized with IMP-6-CPE (Escherichia coli [n = 8], Klebsiella oxytoca [n = 5], Enterobacter cloacae [n = 5], Klebsiella pneumoniae [n = 3] and Klebsiella aerogenes [n = 1]) were identified. There were diverse PFGE profiles and sequence types (STs) in most of the species except for K. oxytoca. All isolates of K. oxytoca belonged to ST29 with similar PFGE profiles, suggesting their clonal transmission. Plasmid analysis by WGS revealed that all 22 isolates but one shared a ca. 50-kb IncN plasmid backbone with blaIMP-6 suggesting interspecies gene transmission, and typing of plasmids explained epidemiological links among cases. A case-control study showed pancreatoduodenectomy, changing drains in fluoroscopy room, continuous peritoneal lavage and enteric fistula were associated with IMP-6-CPE acquisition among the patients. Plasmid analysis of isolates in an outbreak of IMP-6-CPE suggested interspecies gene transmission and helped to clarify hidden epidemiological links between cases.

628. Integrated Surveillance for Antimicrobial-Resistance in Salmonella From Clinical and Retail Meat Sources—Pennsylvania, 2015–2017

BackgroundAntimicrobial resistance (AMR) in foodborne pathogens of animal origin, including nontyphoidal Salmonella (NTS), is a public health concern. Pennsylvania conducts integrated surveillance for AMR in NTS from human and animal sources in collaboration with the FDA and CDC National Antimicrobial Resistant Monitoring System (NARMS).MethodsWe reviewed pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility (SST) and whole-genome sequencing (WGS) data for isolates from animal and food sources, including 96 NTS from 2,520 meat samples (poultry, ground beef and pork chops) purchased during 2015–2017 from randomly selected retail outlets in Pennsylvania. SST to 15 antimicrobial agents was done on 109 NTS clinical isolates that had similar PFGE patterns to meat isolates. SST and WGS were used to characterize all isolates from meat and two clinical isolates from 2017.Results28 (29.2%) and 17 (17.7%) NTS isolated from meat sources were resistant to ≥3 and ≥5 antibiotics classes, respectively. Resistance to ceftriaxone rose from 12% (3/25) in 2015 to 27% (10/37) in 2016 and resistance to amoxicillin/clavulanate also increased. Plasmid-mediated bla CMY-2 β-lactam resistance genes that hydrolyze extended-spectrum cephalosporins (ESC) increased from 12% in 2015 (3/25) to 18.9% (7/37) in 2016. Four blaCTX-M-65 genes that confer resistance to extended-spectrum β-lactamases (ESBLs) were identified in 2016 (n = 3) and 2017. Of the 109 clinical isolates, 25.7% demonstrated resistance to ≥3 and 11% to ≥5 antibiotics classes, respectively. No clinical isolates were resistant to ceftriaxone in 2015, 12.5% (6/48) and 24.3% (9/37) were resistant in 2016 and 2017, respectively. Resistance to amoxicillin/clavulanate was demonstrated in 8.3% (4/48) of isolates in 2016 (figure). Two clinical isolates carried blaCTX-M-65 ESB Ls genes and were resistant to eight antimicrobial agents (ACSSuTCxNalCot. Phenotype).ConclusionNTS (≥25%) from animal and human sources were multidrug-resistant and harbored CMY-2 and CTX-M-65 genes. Dissemination of genes that confer resistance to ESBLs and ESCs in NTS undermines recommended treatment for severe infections and underscores the need for One-Health surveillance and antimicrobial stewardship efforts. Disclosures All authors: No reported disclosures.

Prevalence of meticillin-resistant and -susceptible coagulase-negative staphylococci with the first detection of the mecC gene among cows, humans and manure in Tunisia

In Europe, a novel mecA homologue - mecC (formerly mecALGA251)- has emerged recently in staphylococci from animals, humans and the environment. This paper reports the first occurrence of the mecC gene in Staphylococcus sciuri from cows and manure in Tunisia and Africa. Forty-nine coagulase-negative staphylococci (CNS) were isolated from the milk of cows with mastitis (n=20), manure (n=20) and human nares (n=9), including 16 Staphylococcus equoruim (32.6%), 12 S. xylosus (24.5%), 12 S. sciuri (24.5%), two S. saprophyticus (4.1%), two S. haemolyticus (4.1%), two S. lentus (4.1%), two S. vitulinus (4.1%) and one S. cohnii (2%). CNS from the three origins carried various resistance genes [mecA, blaZ, tet(K), erm(A), erm(B), msr(A)], suggesting an ongoing genetic exchange among CNS from the three niches. The mecA gene was detected in CNS (n=11) recovered from cows, manure and humans, whereas the mecC gene (n=3) was only detected in CNS from cows and manure. Various staphylococcal cassette chromosome mec (SCCmec) - SCCmec type I (n=1), II (n=3), IV (n=2), V/VII (n=2) and untypeable (n=3) - and diverse pulsed-field gel electrophoresis (PFGE) patterns were observed in mecA-positive CNS. Otherwise, similar SCCmec types and PFGE patterns were found in meticillin-resistant CNS within different farms and origins, showing the potential of SCCmec interspecies exchange and circulation of the same clones of meticillin-resistant CNS in the human-animal-environment interface. This study provides baseline data to support clonal dissemination of CNS between cows, humans and manure, and indicates the possible transmission of the mecC gene to humans in contact with cows and manure.

Real world incidence estimation methodologies used for surveillance of HIV in repeat blood donors

This study was performed to characterize CTX-M type extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae carriage in asymptomatic health individuals, which has not been well investigated, in a community of the Okinawa prefecture, Japan. Fecal samples were voluntary collected from asymptomatic healthy individuals who were going to take a routine medical checkup. The collected fecal samples were inoculated on MacConkey agar supplemented with 2 μg/ml of cefotaxime and incubated at 37 °C. Randomly selected three lactose-fermented colonies per each sample were analyzed. Genetic relatedness among the CTX-M type ESBL-producing Enterobacteriaceae isolates were performed by pulsed-field gel electrophoresis (PFGE) after confirmation of ESBL phenotype and determination of bacterial species. Location of blaCTX-M was confirmed by S1-PFGE, I-CeuI-PFGE and the Southern blotting hybridization. ESBL-producing Enterobacteriaceae was isolated from 32 (12.2%) of the collected 263 fecal samples, and 96 ESBL-producing Enterobacteriaceae isolates were obtained. CTX-M type ESBL-producing Escherichia coli B2 were major (67 isolates, 72.0%) and 40 (59.7%) of the 67 CTX-M type ESBL -producing E. coli B2 were E. coli B2-ST131. Three CTX-M type ESBL-producing E. coli B2-ST131 isolates from asymptomatic healthy individuals showed similar PFGE band patterns as five CTX-M type ESBL -producing E. coli B2-ST131 isolates from a hospital locates in the same area of the target community. Chromosomally-transferred blaCTX-M was observed in 10.0% of the examined CTX-M type ESBL-producing Enterobacteriaceae isolates. We report current situation CTX-M type ESBL-producing Enterobacteriaceae carriage in asymptomatic healthy individuals of the Okinawa prefecture, Japan. In addition, our results indicated that worldwide distributed CTX-M type ESBL-producing E. coli B2-ST131 has been spread in a community. Therefore monitoring of ESBL-producing Enterobacteriaceae in healthy individuals is important.

Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide.

PurposePlasmids of the incompatibility group X type 3 (IncX3) were described carrying various carbapenemase genes in carbapenemase-producing Enterobacteriaceae (CPE) worldwide and in the United Arab Emirates (UAE), as well. To understand the driving force behind the emergence of such plasmids in the UAE, the relationship between IncX3 plasmids encountered locally and globally was investigated.MethodsCPE strains isolated in the UAE during 2009–2014 were screened by X3 PCR-based replicon typing. The clonal relationship of CPE carrying IncX3 plasmids was determined by multi-locus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). Complete sequence of selected IncX3 plasmids was determined. Phylogenetic relationship between the carbapenemase carrying IncX3 plasmids from the UAE and of those reported worldwide was established by comparing the plasmid backbones.Results10.2% of the 295 CPE tested were identified to carry IncX3 plasmids: 13 Escherichia coli, 13 Klebsiella pneumoniae, two Enterobacter cloacae, one Citrobacter freundii and one Morganella morganii isolate, respectively. Most of them were non-clonal; with small clusters of triplets and pairs of E. coli and K. pneumoniae, and a cluster of five K. pneumoniae ST11 exhibiting >90% similar PFGE patterns, respectively. The 30 isolates harbored either blaNDM-1, blaNDM-4, blaNDM-5, blaNDM-7, blaOXA-181 or blaKPC-2 carbapenemase genes on IncX3 plasmids. Phylogenetic analysis of the backbone region of IncX3 plasmids carrying various beta-lactamase genes from the UAE (n=23) and that of North-America, Europe, Asia and Australia (n=35) revealed three clusters based on the carbapenemase genes carried: plasmids harboring blaOXA-181 and blaNDM-5 formed two distinct groups, whereas backbones of plasmids with blaNDM-1, blaNDM-4 and blaNDM-7 clustered together. Each cluster contained plasmids of diverse geographical origin.ConclusionThe findings suggest that different carbapenemase gene carrying IncX3 plasmids encountered in the UAE do not evolve locally, rather are subtypes of this epidemic plasmid emerging in this country due to international transfer.

Investigation of a hospital Enterobacter cloacae NDM-1 outbreak using whole genome sequencing

Caprbapenemase-producing Gram-negative micro-organisms are emerging as a major clinical problem. The infections caused by these highly resistant and hospital-adapted pathogens may become untreatable using existing antibiotics. Over a three year period, six patients at a large UK tertiary-referral hospital were colonised or infected with carbapenem-resistant Enterobacter cloacae carrying the blaNDM-1 metallo-β-lactamase gene. Environmental isolates were also obtained from a clinical wash-hand basin and taps. The isolates had very similar pulsed-field gel electrophoresis profiles, suggesting they were related, although only four of the cases had epidemiological links. Whole genome sequencing showed the isolates had the same genomic background (sequence type ST114). Genes encoding seven different extended-spectrum and inhibitor-resistant β-lactamase and carbapenemase enzymes (blaNDM-1; blaCTX-M-15; blaACT-16; blaVEB-1; blaTEM-1; blaOXA-1 and blaOXA-10) were present, in addition to multiple genes and mutations conferring resistance to aminoglycosides, quinolones, trimethoprim, tetracycline, sulphonamide, chloramphenicol, rifampicin and fosfomycin. Phenotypic testing indicated sensitivity only to colistin and tigecycline. Genome-wide single nucleotide polymorphism analysis showed the four linked isolates were closely related, and differed from the unlinked isolates by 16–24 SNPs. Moreover, resistance encoding plasmids had been lost in the two unlinked isolates. This suggested these isolates, although sharing a recent common ancestor, had evolved in different environments. Whole genome sequencing allowed resolution of very closely related E. cloacae strains, and confirmed the outbreak did not extend beyond the linked patients. Sequencing also confirmed the same highly resistant E. cloacae strain had persisted within a clinical unit for over two years, despite rigorous efforts to eradicate it.

Characterization of Antimicrobial Resistance in Serratia spp. and Citrobacter spp. Isolates from Companion Animals in Japan: Nosocomial Dissemination of Extended-Spectrum Cephalosporin-Resistant Citrobacter freundii.

In many countries including Japan, the status of emerging antimicrobial resistance among Serratia spp. and Citrobacter spp. in companion animals remains unknown because these genera are rarely isolated from animals. In this study, 30 Serratia spp. and 23 Citrobacter spp. isolates from companion animals underwent susceptibility testing for 10 antimicrobials. Phenotypic and genetic approaches were used to identify the mechanisms of extended-spectrum cephalosporins (ESC). Subsequently, ESC-resistant Citrobacter spp. strains underwent multilocus sequence typing and pulsed-field gel electrophoresis (PFGE). A significantly higher rate (34.8%) of ESC resistance was observed in Citrobacter spp. isolates than in Serratia spp. isolates (0%). ESC resistance was detected in five C. freundii strains, two C. portucalensis strains, and one C. koseri strain. All of the ESC-resistant Citrobacter spp. strains harbored CMY-type and/or DHA-type AmpC β-lactamases. Three C. freundii strains harbored the CTX-M-3-type extended-spectrum β-lactamases. Notably, the three blaCTX-3-producing and two blaCMY-117-bearing C. freundii strains (obtained from different patients in one hospital) had the same sequence type (ST156 and ST18, respectively) and similar PFGE profiles. We believe that ESC-resistant Citrobacter spp. are important nosocomial pathogens in veterinary medicine. Therefore, infection control in animal hospitals is essential to prevent dissemination of these resistant pathogens.

Neonatal sepsis due to glycopeptide resistant Enterococcus faecium from colonized maternal gut- rare case evidence

BackgroundVancomycin-resistant enterococcal infections in the neonatal ICU are growing global problems. We report a case of neonatal septicemia by multidrug-resistant vancomycin-resistant Enterococcus faecium (VRE), the source of infection being the mother’s gut.Case presentationA newborn male child admitted to the neonatal intensive care unit (NICU) was diagnosed to have mild meconium aspiration syndrome, early onset neonatal septicemia, and bacteremia by multidrug and vancomycin-resistant Enterococcus faecium. Screening of gut flora of the baby and the mother were carried out to trace the source of infection. Stool cultures of the mother and the baby yielded Vancomycin-Resistant Enterococcus faecium. All three isolates of Enterococcus faecium had similar antibiogram, harbored the vanA gene and similar pulsed-field gel electrophoresis pattern. Baby responded to the 1 week therapy with oral linezolid suspension 20 mg/kg/day, 1 ml/t.d.s. No VRE was isolated from baby on a repeat stool culture 1 week after the linezolid therapy. He was discharged with the advice for the continuance of linezolid for seven more days.ConclusionIsolation of MDR-VRE from the blood culture of the baby and stool specimens of the mother and the baby with the same antibiogram profile and clonal similarities reveals that maternal gut colonization was responsible for neonatal sepsis. Optimal infection control measures and the development of guidelines for monitoring VRE colonization in pregnant women might be useful in reducing the occurrence of neonatal sepsis.

1201. A Prolonged Multispecies Outbreak of Carbapenemase-Producing Enterobacteriaceae Due to Transmissible Plasmid With Carbapenemase Gene

BackgroundIn 2010, a multispecies outbreak of IMP type carbapenemase-producing Enterobacteriaceae (IMP-CPE) occurred at a large acute care hospital in Japan. The outbreak continued for years involving more than 100 patients mainly in surgical wards.MethodsBecause of the long period of the outbreak, investigation were focused on hospitalized patients whose clinical samples were positive for IMP-CPE between July 2013 and March 2014. A case–control study was conducted for cases who underwent abdominal surgery with controls from whom meropenem-susceptible Enterobacteriaceae were isolated. Pulsed-field gel electrophoresis (PFGE) was used for molecular typing. To evaluate genetic relationship among IMP-CPE isolates of different species, plasmid analysis using S1 nuclease to separate plasmid and chromosomal DNA followed by plasmid DNA extraction and whole-genome sequencing (WGS) was conducted.ResultsDuring the study period, 22 cases were identified and 22 IMP-CPE isolates which consisted of eight Escherichia coli, five Klebsiella oxytoca, five Enterobacter cloacae, three Klebsiella pneumoniae and one Enterobacter aerogenes were obtained. All five isolates of K. oxytoca had similar PFGE profiles which suggested clonal transmission. However, PFGE profiles of E. coli, E. cloacae and K. pneumoniae isolates were diverse. Plasmid analysis revealed that all 22 isolates shared ca. 50 kb IncN plasmid with blaIMP-6 which implies interspecies transmission of it The case–control study which adjusted by days of hospitalization with 11 cases and 24 controls revealed that pancreato-duodenectomy (adjusted odds ratio (aOR) = 6.4, 95% confidence interval (CI) 1.3–32.4) and enteric fistula (aOR = 8.0, 95% CI 1.5–41.9) were associated with IMP-CPE acquisition. Use of endoscopy within the past six months was not associated with IMP-CPE (aOR = 0.8 95% CI 0.2–4.2). With a bundled infection control with Osaka City Public Health Office, the outbreak was contained in July 2016.ConclusionDissemination of carbapenemase gene by transmissible plasmid can play a critical role to complicate epidemiology of CPE outbreak and made it difficult to control. Plasmid analysis using WGS technology is a promising tool to untangle it.Disclosures All authors: No reported disclosures.