Antimicrobial Resistance and PFGE Molecular Typing of Salmonella enterica Serovar Gallinarum Isolates from Chickens in South Korea from 2013 to 2018

Antimicrobial resistance and pulsed-field gel electrophoresis (PFGE) genotypes of collected S. enterica ser. Gallinarum isolates were investigated to examine the epidemiological relationship between field outbreak isolates of S. enterica ser. Gallinarum. Thirty S. enterica ser. Gallinarum isolates collected from poultry farms with FT outbreaks from 2013 to 2018 in South Korea were analyzed. All isolates were resistant to at least 3 of the 18 antimicrobials tested and exhibited an MDR phenotype. All isolates showed resistance to streptomycin, sulfisoxazole, and colistin. One isolate was resistant to 9 antimicrobials. The antimicrobial resistance profile, streptomycin-sulfisoxazole-colistin-nalidixic acid-ciprofloxacin-gentamicin (18/30, 60.0%), was the most prevalent. PFGE types were classified into 10 groups with a 100% correlation cutoff in dendrograms for 30 field isolates. The dominant PFGE types were 1 (8/30, 26.7%), 4 (7/30, 23.3%), and 9 (5/30, 16.7%). Interestingly some isolates collected from the same and different companies had the same PFGE type. We reported a high MDR rate in S. enterica ser. Gallinarum isolates. The present study highlights the occurrence of horizontal spread and cyclic contamination of MDR S. enterica ser. Gallinarum within the same company. Furthermore, we showed cross-contamination between different companies. The characterization of these isolates would be helpful in the development of prevention and control strategies for MDR S. enterica ser. Gallinarum infection in South Korea.

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing

Background and Objectives: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. Materials and Methods: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4% for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. Results: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; І) PFGE type of B. mallei Razi 325 strain, ІІ) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ІІІ) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. Conclusion: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies.

Emergence of a mupirocin-resistant, methicillin-susceptible Staphylococcus aureus clone associated with skin and soft tissue infections in Greece

Background Staphylococcus aureus causes various infections, including skin and soft tissue infections (SSTIs). In this study, methicillin-susceptible S. aureus (MSSA) from SSTIs among patients in three tertiary-care hospitals in Greece were studied in terms of antimicrobial resistance, clonal distribution, toxin and adhesin genes carriage. Results During a five-year period (2014–2018), 6145 S. aureus were recovered from 13,244 patients with SSTIs and tested for antimicrobial susceptibility. MSSA were 4806 (78.21 %) including 1484 isolates with mupirocin minimum inhibitory concentration (MIC) > 64 mg/L (30.88 %). Two hundred and sixty representative mupirocin-resistant MSSA were analyzed for genes encoding Panton-Valentine leukocidin (PVL, lukS/lukF-PV), exfoliative toxins (eta, etb), adhesin FnbA (fnbA) and resistance genes mupA (high-level resistance to mupirocin), fusB (fusidic acid), aminoglycosides’ modifying enzymes, ermA, ermC and msrA (macrolides/lincosamides) by PCRs. Strains were classified into clones by PFGE and MLST. All mupirocin-resistant MSSA were penicillin-resistant; 92.7 % expressed resistance to fusidic acid and 88.9 % to tobramycin. All 260 molecularly analyzed isolates were mupA-positive; all fusidic acid-resistant (241/260) carried fusB whereas, the tobramycin-resistant ones (230), ant(4′)-Ia. The majority carried eta (93.85 %), etb (98.08 %) and fnbA (88.85 %). PFGE typing revealed a mostly unvarying population; 260 MSSA were grouped into three types. One major eta/etb-positive clone comprising of 258/260 strains (99.2 %), PFGE type 1, was classified as ST121, including nine strains co-carrying PVL. Another PVL-positive strain was identified as ST1, and one toxins-negative as ST21. Conclusions A mupirocin-resistant MSSA clone, ST121, carrying resistance, exfoliative toxins and adhesin genes, was spread and predominated in SSTIs from patients in Greece during the five-year studied period.

The Changing Landscape of Molecular Epidemiology of Staphylococcus Aureus Infections in Children.

Background: Staphylococcus aureus infections cause significant morbidity and mortality in children and adolescents. Aim of this study was to investigate the molecular epidemiology and antibiotic resistance of Staphylococcus aureus clinical isolates from children and adolescents.Methods: All S. aureus isolates recovered from patients aged < 18 years, admitted to a referral hospital, with culture-proven invasive or non-invasive, community-associated or community-onset healthcare-associated or hospital-associated infections during the 4-year period from January 2015 to December 2018 were analyzed for antimicrobial resistance, virulence genes, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST).Results: Among 139 S. aureus clinical isolates, 16 (11.5%) were methicillin-resistant (MRSA) and 123 (88.5%) methicillin-susceptible (MSSA). MSSA infections increased significantly over time (2017-2018 vs. 2015-2016, 0R 3.32; 95%CI 1.18-8.96; p 0.03) along with increasing resistance to fusidic acid (OR 2.38; 95%CI 1.14-5.12; p 0.02) and in staphylococcal scalded skin syndrome prevalence (OR 3.24; 95% CI 1.10-8.36; p 0.03). A total of five sequence types (ST) were identified among 58 isolates that were analyzed by MLST. By PFGE typing, 22 pulsotypes were identified, whereas, PFGE type 1 classified as ST121 clone was the predominant (40/58,68.9%). MRSA were distributed into four pulsotypes and PFGE type C- ST80 was the most frequent. ST121 strains carried fnbA (40/40), eta/etb genes (29/40) and lukF/S-PVL genes in 3/40 cases. All ST121 exhibited high resistance percentage to fusidic acid and were increasingly resistant to mupirocin. Conclusion: In our population, a CA-MSSA clone emerged, resistant to fusidic acid and increasingly resistant to mupirocin which belonged to the PFGE type 1, ST121 clone, harbored exfoliative toxins genes and was associated with rising trends of SSSS.

Investigation of Commensal Escherichia coli Populations of Cormorant Hatchlings in the Absence of Anthropogenic Impacts in Remote Areas of West Mongolia

To increase our understanding of bacterial intestinal colonization in animal populations lacking substantial anthropogenic influence we studied the diversity of E. coli in cormorants from the pristine West-Mongolian steppe. E. coli were isolated from individual birds of two cormorant colonies located on small islands in lakes at least 100 km away from human settlements. Diversity of the isolates was studied using pulsed-field gel electrophoresis (PFGE). 137 isolates of cormorant colony-1 and 75 isolates of cormorant colony-2 resulted in 60 and 33 PFGE types, respectively. Representative strains of each PFGE type were analyzed via PCR in terms of phylogroups and extraintestinal virulence-associated genes (exVAGs). Bacterial adhesion to the chicken intestinal cell line CHIC-8E11 and antimicrobial resistance was also determined. Most isolates belonged to phylogroup B1 (68.3%) followed by B2 and E with B2 harboring the highest total number of exVAGs per isolate. Unexpectedly, a PFGE type with relatively few exVAGs displayed the highest isolation frequency, also showing a high adhesion rate. Comparative analysis of exVAGs to other E. coli populations of wildlife origin revealed that the secreted autotransporter toxin encoding sat gene was only present in cormorants. Overall, E. coli in cormorants maintained a high diversity under minimal anthropogenic influences, which likely enables intestinal colonization.

Co-Occurrence of Listeria spp. and Spoilage Associated Microbiota During Meat Processing Due to Cross-Contamination Events

A large part of foodborne outbreaks related to Listeria monocytogenes are linked to meat and meat products. Especially, recontamination of meat products and deli-meat during slicing, packaging, and repackaging is in the focus of food authorities. In that regard, L. monocytogenes persistence in multi-species biofilms is one major issue, since they survive elaborate cleaning and disinfection measures. Here, we analyzed the microbial community structure throughout a meat processing facility using a combination of high-throughput full-length 16S ribosomal RNA (rRNA) gene sequencing and traditional microbiological methods. Samples were taken at different stages during meat cutting as well as from multiple sites throughout the facility environment to capture the product and the environmental associated microbiota co-occurring with Listeria spp. and L. monocytogenes. The listeria testing revealed a widely disseminated contamination (50%; 88 of 176 samples were positive for Listeria spp. and 13.6%; 24 of 176 samples were positive for L. monocytogenes). The pulsed-field gel electrophoresis (PFGE) typing evidenced 14 heterogeneous L. monocytogenes profiles with PCR-serogroup 1/2a, 3a as most dominant. PFGE type MA3-17 contributed to the resilient microbiota of the facility environment and was related to environmental persistence. The core in-house microbiota consisted mainly of the genera Acinetobacter, Pseudomonas, Psychrobacter (Proteobacteria), Anaerobacillus, Bacillus (Firmicutes), and Chryseobacterium (Bacteroidota). While the overall microbial community structure clearly differed between product and environmental samples, we were able to discern correlation patterns regarding the presence/absence of Listeria spp. in both sample groups. Specifically, our longitudinal analysis revealed association of Listeria spp. with known biofilm-producing Pseudomonas, Acinetobacter, and Janthinobacterium species on the meat samples. Similar patterns were also observed on the surface, indicating dispersal of microorganisms from this multispecies biofilm. Our data provided a better understanding of the built environment microbiome in the meat processing context and promoted more effective options for targeted disinfection in the analyzed facility.

Analysis of Benzalkonium Chloride Resistance and Potential Virulence of Listeria monocytogenes Isolates Obtained from Different Stages of a Poultry Production Chain in Spain

ABSTRACT Listeria monocytogenes can survive in food production facilities and can be transmitted via contamination of food during the various stages of food production. This study was conducted to compile the results of three independent previous studies on the genetic diversity of L. monocytogenes in a poultry production company in Spain and to determine the potential virulence and sanitizer resistance of the strains by using both genotype and phenotype analyses. L. monocytogenes was detected at three production stages: a broiler abattoir, a processing plant, and retail stores marketing fresh poultry products from the same company. These three stages spanned three locations in three provinces of Spain. A set of 347 L. monocytogenes isolates representing 39 subtypes was obtained using pulsed-field gel electrophoresis (PFGE). A total of 28 subtypes (68%) had a full-length internalin A gene, and two subtypes had a phenotype with low potential for virulence because of a mutation in the prfA gene. A total of 32 subtypes (82%) were classified as benzalkonium chloride resistant (BAC-R) and contained the resistance determinant bcrABC (21 subtypes, 54%) or the resistance gene qacH (11 subtypes, 28%). A total of 13 persistent BAC-R subtypes (minimum of 3 months between the first and last sample from with the isolate was recovered) were identified at the abattoir and processing plant. The three production stages shared a unique subtype (PFGE type 1), which had the mutation in the prfA gene and the bcrABC resistance determinant. Whole genome sequencing revealed this subtype to be sequence type 31. Limited genetic diversity was noted in the isolates studied, including some subtypes that were persistent in the environment of the investigated facilities. Given the high prevalence of BAC-R subtypes, these results support the association between resistance to biocides and persistence of L. monocytogenes. HIGHLIGHTS

561. Genomic Epidemiology of Methicillin-Resistant Staphylococcus aureus in Two Cohorts of High-Risk Military Trainees

Background Methicillin-resistant Staphylococcus aureus (MRSA) skin and soft-tissue infections (SSTIs) are common among military recruits. Identifying which strains are responsible for SSTI and understanding the underlying transmission dynamics is critical to developing appropriate interventions for this high-risk population. Methods A cohort study of US Army Infantry trainees at Fort Benning, GA (June and September 2015). Participants from two training Companies were screened for colonization on multiple anatomic sites throughout the 14-week cycle as well as the time of clinical infection. MRSA+ samples were sequenced with Illumina HiSeq. Multi-locus sequence type (MLST) and virulence genes were identified in silico. Single nucleotide polymorphism (SNP) distances between soldiers’ bacteria were compared with assessing for potential transmission. Results Of 383 soldiers enrolled, 84 (22%) were colonized with MRSA during the study. Forty-two of 84 had a single positive colonization sample, of which 76% were from anatomical sites other than the nares (36% oropharyngeal, 26% perianal, 14% inguinal). Twelve trainees had MRSA SSTI during training (50% had colonization detected prior to or at infection). All were PFGE-type US300 (ST8) and were lukS/lukF-positive. SNP-based phylogenetic analyses and epidemiologic data indicate that most MRSA positives at baseline were due to unique importations from various community origins, suggesting that the ongoing MRSA epidemic is not due to a single endemic strain circulating on base. Following importation, extensive transmission then occurred, with multiple STs implicated. Transmission appeared restricted to within Companies, and predominantly within platoons. Conclusion Frequent colonization at baseline suggests a need for extensive MRSA screening and decolonization upon arrival to base, followed by ongoing infection control measures throughout training to prevent recolonization/infection. As multiple anatomical sites appear to play a role in transmission of MRSA, this may have important implications for screening protocols and control both in community and hospital-based settings. Disclosures All authors: No reported disclosures.

The Washing Machine as a Reservoir for Transmission of Extended-Spectrum-Beta-Lactamase (CTX-M-15)-Producing Klebsiella oxytoca ST201 to Newborns

ABSTRACT During the period from April 2012 to May 2013, 13 newborns (1 to 4 weeks of age) and 1 child in a pediatric hospital ward in Germany were colonized with Klebsiella oxytoca producing an extended-spectrum beta-lactamase (ESBL) (CTX-M-15). A microbiological source-tracking analysis with human and environmental samples was carried out to identify the source and transmission pathways of the K. oxytoca clone. In addition, different hygienic intervention methods were evaluated. K. oxytoca isolates were detected in the detergent drawer and on the rubber door seal of a domestic washer-extractor machine that was used in the same ward to wash laundry for the newborns, as well as in two sinks. These strains were typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The environmental findings were compared with those for the human strains and the isolates detected on clothing. The results from both techniques showed that the strains were identical (sequence type 201 and PFGE type 00531, a clone specific to this hospital and not previously isolated in Germany), emphasizing the washing machine as a reservoir and fomite for the transmission of these multidrug-resistant bacteria. After the washing machine was taken out of use, no further colonizations were detected during the subsequent 4-year period. IMPORTANCE Washing machines should be further investigated as possible sites for horizontal gene transfer (ESBL genes) and cross-contamination with clinically important Gram-negative strains. Particularly in the health care sector, the knowledge of possible (re-)contamination of laundry (patients’ clothes and staff uniforms) with multidrug-resistant Gram-negative bacteria could help to prevent and to control nosocomial infections. This report describes an outbreak with a single strain of a multidrug-resistant bacterium (Klebsiella oxytoca sequence type 201) in a neonatal intensive care unit that was terminated only when the washing machine was removed. In addition, the study implies that changes in washing machine design and processing are required to prevent accumulation of residual water where microbial growth can occur and contaminate clothes.

Prevalence, Antimicrobial Resistance, and Molecular Typing of Thermophilic Campylobacter Spp. in a Greek Poultry Slaughterhouse

Campylobacter species are one of the leading causes of foodborne disease. Poultry is a major reservoir and source of its transmission to humans. The aim of this study was to estimate the prevalence and antimicrobial resistance of Campylobacter spp. isolated from chicken carcasses, the environment, and processing equipment of a poultry slaughterhouse in Greece, to identify the dominant Campylobacter species and to determine if there are clonal relationships among the isolates. Fifty poultry samples and 25 environmental samples were examined using microbial cultures and PCR. Forty-nine of 50 poultry samples (98%) were found to be positive for Campylobacter spp. The environment of the slaughterhouse was also found to be significantly contaminated with Campylobacter spp. Thirty-seven isolates were found to be susceptible to all antimicrobials tested (56.1%) and 29 isolates showed resistance to at least two of the antimicrobials tested (43.9%). We observed 24 different PFGE-types among the 53 isolates with 14 of them isolated only once, while five PFGE-types were represented by two isolates. The remaining 29 isolates were represented by five PFGE-types each consisting of three to 12 isolates. Regarding the relationship of the PFGE types and corresponding resistance profiles, all strains of each PFGE-type shared the same antimicrobial resistance profile. This study reports evidence for Campylobacter spp. cross-contamination among broiler carcasses in a Greek slaughterhouse.