Pulsed-field Gel Electrophoresis Typing Research Articles

The prevalence and characteristics of extended-spectrum β-lactamase Escherichia coli in raw milk and dairy farms in Northern Xinjiang, China

In this study, extended-spectrum β-lactamase (ESBL) Escherichia coli were isolated from five dairy farms in three areas of northern Xinjiang, China. Molecular biological techniques were used to systematically analyze drug resistance phenotypes and genotypes, virulence genes, phylogenetics, biofilm formation (BF), and pulsed field gel electrophoresis (PFGE) typing of isolated ESBL E. coli strains. A total of 766 samples were collected from five dairy farms in Shihezi, Urumqi and Yili, from which 149 (19.5 %, 95 % CI: 16.65 %–22.25 %) ESBL E. coli strains were isolated. Their distribution and contamination levels varied from region to region, with 16.2 % (68/419) in Urumqi, 22.4 % (60/268) in Shihezi, and 26.6 % (21/79) in Yili. The majority of isolates (97.3 %, 145/149) harbored the β-lactamase blaCTX-M gene; while blaCTX-M-1 was the dominant phylogenetic group. The analysis of 21 resistance genes and the susceptibility to 13 different antibiotics showed that 91.3 % (136/149) of strains were resistant to three or more antibiotics. Thirty-six strains (24.2 %) belonged to extraintestinal pathogenic E. coli (ExPEC), and phylogenetic typing results were mainly grouped A (50.3 %) and B1 (37.6 %). Also, the biofilm assay revealed that 112 strains (75.2 %) could form biofilms. PFGE results showed that the 49 isolates revealed 21 major pulsotypes (P1–P21) and 28 subtypes with 80 % similarity, indicating the overall genetic diversity in the distribution area and sources of the samples.

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.

Biofilm formation, interaction and survival within A549 pneumocytes of Klebsiella pneumoniae clinical strains: identification of pulsotypes, multidrug-resistance and genes coding for adhesins

Klebsiella pneumoniaehas become one of the major causes of hospital-acquired infections over decades due to the spread of virulent clones harboring resistant genes to multiple antimicrobial agents. The aim of this study was to investigate phenotypic and genotypic features of virulence mechanism expressed by K. pneumoniae clinical isolates of different PFGE types, including biofilm formation, interaction with pneumocytes A549 lineage and experimental infection by using C. elegans nematodes. A total of 17 K. pneumoniae strains were isolated from different clinical specimens including blood, urine and respiratory infections. In this present study, 11 strains presented a varied multidrug-resistance profile harboring resistance genes coding for betalactams, aminoglicosydes, fluorquinolones and carbapenemases. PFGE analysis demonstrated the presence of four distinct pulsotypes among K. pneumoniae strains harboring virulence genes for siderophores and fimbiae type 1 and type 3. High adherence and biofilm formation were positively correlated for both polystyrene and glass surfaces in all K. pneumoniae strains analyzed. K. pneumoniae clinical strains showed the ability of adherence, internalization and persistence within human pulmonary epithelial A549 cell line, at different levels. Respiratory infections demonstrated a higher heterogeneity of PFGE types and levels of adherence, intracellular survival and persistence.K. pneumoniae strains were also submitted to Carnohabidits elegans in vivo infection model and data showed that after 24 hr almost 10% of urine-culture isolates worms were dead evidencing virulence profile. Notably, K. pneumoniae strains, presenting virulence genes, was significantly more virulent than those who did not presented any virulence gene after 5 days (survival >60% and >40%).

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

ABSTRACTPathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries.IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

Emerging resistance to ceftriaxone treatment owing to different ampD mutations in Enterobacter roggenkampii

The Enterobacter cloacae complex is responsible for a variety of infections in hospitalized patients and is resistant to β-lactam antibiotics owing to the expression of AmpC β-lactamase. We report emerging resistance in Enterobacter roggenkampii exposed to ceftriaxone and explore the mechanism underlying mutations responsible for this resistance. Three strains were derived from different samples from one patient (blood and liver abscess fluid). Antimicrobial susceptibility was evaluated by standard broth microdilution, while ampC expression was determined via RT-PCR. Genetic relatedness was evaluated via pulsed-field gel electrophoresis (PFGE). Species identification and comparative genome analysis were performed via genome sequencing. Mutation rate testing and selection of AmpC-derepressed mutants were conducted to explore the mutation mechanism. E. roggenkampii F1247 was susceptible to third-generation cephalosporins (3GCs); F95 and F1057, found in blood sample on day 11 and liver abscess drainage fluid on day 25, were resistant. ampC expression was 341- and 642-fold higher in F95 and F1057, respectively, than in F1247. Three isolates were the same PFGE and sequence types (ST1778) and were highly homologous (2 and 4 core genome single nucleotide polymorphism differences). Compared to F1247, F95 possessed a 575bp deletion, including 537bp of ampD, whereas F1057 harbored only one amino acid mutation (Leu140Pro in ampD). The mutation rates from F1247 exposure to cefotaxime, ceftazidime, ceftriaxone, piperacillin-tazobactam, and cefepime were (1.90±0.21)×10-8, (3.18±0.43)×10-8, (2.00±0.20)×10-8, (2.92±0.29)×10-9, and zero, respectively. In vitro-selected mutations responsible for resistance were identified in ampD, ampR, and dacB. E. roggenkampii may develop resistance in vivo and in vitro upon exposure to 3GCs and to a lesser extent to piperacillin-tazobactam. 3GCs should not be used as a monotherapy for E. roggenkampii infections. Therapy using cefepime or carbapenems may be preferred to piperacillin-tazobactam in the treatment of E. roggenkampii, especially if source control is difficult.

Genetic Diversity, Antimicrobial Resistance and Survival upon Manure Storage of Campylobacter jejuni Isolated from Dairy Cattle Farms in the Cantabric Coast of Spain

The aim of this study was the examination of the genetic diversity and antimicrobial susceptibility of C. jejuni isolates obtained from dairy farms situated in Cantabria. The presence of Campylobacter jejuni was scrutinized in dairy farms situated in the Cantabria region (Atlantic coast, North of Spain). A total of 520 samples were collected from 12 dairy farms and 62 C. jejuni isolates were achieved. Sixty-one (61) of the isolates proceeded from fresh feces and only one from the stable (soil). Characterization of the isolates was conducted by Pulsed Field Gel Electrophoresis (PFGE) analysis. Antimicrobial susceptibility testing was carried out by standardized disk diffusion test. The PFGE analysis showed a high genetic diversity. From the 62 C. jejuni isolates, 27 different PFGE types were obtained with 70% similarity. The results of the antimicrobial susceptibility tests showed that 21 out of 27 strains were resistant to ciprofloxacin (78%), and 15 of them were also resistant to tetracycline (55%), whereas none of the 27 strains analyzed were resistant to erythromycin. C. jejuni was capable of surviving in livestock waste for at least 20–25 days, whereas the maximum detectable survival time on crops was of six days. This study reveals the high genetic diversity and ciprofloxacin resistance of C. jejuni in dairy cattle farms in Northern Spain, a fact that highlights the urgent need for the surveillance and control of this foodborne pathogen.

Characterization of NDM-5 Carbapenemase-Encoding Gene (bla NDM-5) - Positive Multidrug Resistant Commensal Escherichia coli from Diarrheal Patients.

PurposeThe multidrug resistance Enterobacteriaceae cause many serious infections resulting in prolonged hospitalization, increased treatment charges and mortality rate. In this study, we characterized blaNDM-5-positive multidrug resistance commensal Escherichia coli (CE) isolated from diarrheal patients in Kolkata, India.MethodsThree CE strains were isolated from diarrheal stools, which were negative for different pathogroups of diarrheagenic E. coli (DEC). The presence of carbapenemases encoding genes and other antimicrobial resistance genes (ARGs) was detected using PCR. The genetic arrangement adjoining blaNDM-5 was investigated by plasmid genome sequencing. The genetic relatedness of the strains was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) methods.ResultsIn addition to colistin, the blaNDM-5-positive CE strains showed resistance to most of the antibiotics. Higher MICs were detected for ciprofloxacin (>32 mg/L) and imipenem (8 mg/L). Molecular typing revealed that three CE strains belonged to two different STs (ST 101 and ST 648) but they were 95% similar in the PFGE analysis. Screening for ARGs revealed that CE strains harbored Int-1, blaTEM, blaCTX-M3, blaOXA-1, blaOXA-7, blaOXA-9, tetA, strA, aadA1, aadB, sul2, floR, mph(A), and aac(6´)-Ib-cr. In conjugation experiment, transfer frequencies ranged from 2.5×10−3 to 8.4x10−5. The blaNDM-5 gene was located on a 94-kb pNDM-TC-CE-89 type plasmid, which is highly similar to the IncFII plasmid harboring an IS26-IS30-blaNDM-5-bleMBL-trpF-dsbd-IS91-dhps structure.ConclusionTo the best of our knowledge, this is the first report on carbapenem resistance involving the blaNDM-5 gene in CE from diarrheal patients. The circulation of blaNDM-5 gene in CE is worrisome, since it has the potential to transfer blaNDM-5 gene to other enteric pathogens.

Investigation of a cluster of Bacillus cereus bacteremia in neonatal care units.

Bacillus cereus is a well-known pathogen for self-limited foodborne illness, and rarely an opportunistic pathogen associated with invasive infections among immunocompromised patients. Nosocomial outbreaks have been rarely reported. Between August and November 2019, four preterm neonates in neonatal care units of a medical center developed late-onset B. cereus bacteremia. An investigation was carried out. Forty-eight environmental specimens were obtained from these neonatal units, skin surface and environmental objects of Patient 4 for the detection of this organism 19 days after the onset of illness of Patient 4. B. cereus isolates from Patient 4, five unrelated patients and environmental objects if identified were further characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). All four infants survived after vancomycin-containing treatment. Patient 4 developed diffuse cerebritis, brain abscess with severe neurologic sequelae. Of the 48 environmental samplings, 26 specimens showed positive for B. cereus, with one major clone (sequence type 365) accounting for 73%. The isolate from Patient 4 (ST427) was identical to one isolate collected from environmental objects in the same unit. After extensive cleaning of the environment and re-institution of the sterilization procedure of hospital linens, which was ceased since two months before the outbreak, no more cases was identified in these units for at least one year. We documented a cluster of B. cereus bacteremia involving four preterm infants, which might be associated with cessation of the procedure for linen sterilization and was successfully controlled by re-institution of this procedure.

Higher In Vivo Fecal Concentrations of Clostridioides difficile Toxins A and B in Patients With North American Pulsed-Field Gel Electrophoresis Type 1/Ribotype 027 Strain Infection.

Ultrasensitive, quantitative Clostridioides difficile stool toxin measurement demonstrated significantly higher concentrations of toxins A and B in patients infected with the North American pulsed-field gel electrophoresis type 1/ribotype 027 (NAP-1/027) strain compared with other strains, providing in vivo confirmation of the in vitro association between NAP-1/027 and elevated toxin production.

Prevalence and Persistence of Multidrug-Resistant Yersinia enterocolitica 4/O:3 in Tonsils of Slaughter Pigs from Different Housing Systems in Croatia.

Yersinia enterocolitica is one of the priority biological hazards in pork inspection. Persistence of the pathogen, including strains resistant to antimicrobials, should be evaluated in pigs from different housing systems for risk ranking of farms. In this 2019 study, tonsils were collected from 234 pigs, of which 69 (29.5%) were fattened on 3 big integrated farms, 130 (55.5%) on 10 medium-sized farms, and 35 (15%) on 13 small family farms. In addition, 92 pork cuts and minced meat samples from the same farms were tested for the presence of Y. enterocolitica using the culture method. Phenotypic and genetic characteristics of the isolates were compared with previously collected isolates from 2014. The overall prevalence of Y. enterocolitica in pig tonsils was 43% [95% CI 36.7–49.7]. In pigs from big integrated, medium-sized, and small family farms, the prevalence was 29%, 52%, and 40%, respectively. All retail samples of portioned and minced pork tested negative for pathogenic Y. enterocolitica, likely due to high hygienic standards in slaughterhouses/cutting meat or low sensitivity of culture methods in these matrices. The highest recovery rate of the pathogen from tonsils was found when alkali-treated PSB and CIN agar were combined. The biosecurity category of integrated and medium farms did not affect the differences in prevalence of Y. enterocolitica (p > 0.05), in contrast to family farms. Pathogenic ail-positive Y. enterocolitica biotype 4 serotype O:3 persisted in the tonsils of pigs regardless of the type of farm, slaughterhouse, and year of isolation 2014 and 2019. PFGE typing revealed the high genetic concordance (80.6 to 100%) of all the Y. enterocolitica 4/O:3 isolates. A statistically significant higher prevalence of multidrug-resistant Y. enterocolitica 4/O:3 isolates was detected in the tonsils of pigs from big integrated farms compared to the other farm types (p < 0.05), with predominant and increasing resistance to nalidixic acid, chloramphenicol, and streptomycin. This study demonstrated multidrug resistance of the pathogen in pigs likely due to more antimicrobial pressure on big farms, with intriguing resistance to some clinically relevant antimicrobials used in the treatment of yersiniosis in humans.

Dissecting Listeria monocytogenes Persistent Contamination in a Retail Market Using Whole-Genome Sequencing.

ABSTRACTListeria monocytogenes is a foodborne pathogen that can cause invasive disease with high mortality in immunocompromised individuals and can survive in a variety of food-associated environments for a long time. L. monocytogenes clonal complex (CC) 87 is composed of ST87 and three other STs and has been identified as the most common subgroup associated with both foods and human clinical infections in China. Therefore, the persistence of CC87 L. monocytogenes in food-associated environments poses a significant concern for food safety. In this study, 83 draft genomes of CC87 L. monocytogenes, including 60 newly sequenced genomes, were analyzed with all isolates from our previous surveillance in Zigong, Sichuang, China. Sixty-eight of the studied isolates were isolated from one retail market (M1 market), while the others were from seven other markets (M2–M8 markets) in the same city. Whole-genome multilocus sequence typing (wg-MLST) and the whole-genome single nucleotide polymorphism (wg-SNP) analysis were performed. Three persistent contamination routes were identified in the M1 market, caused by 2 clusters (A and B) and a wgST31 type. Cluster A isolates were associated with the persistent contamination in a raw meat stall (M1-S77), while Cluster B isolates caused a persistent contamination in aquatic foods stalls. Five wgST31 isolates caused persistent contamination in a single aquatic stall (M1-S65). A pLM1686-like plasmid was found in all Cluster A isolates. A novel plasmid, pLM1692, a truncated pLM1686 plasmid without the cadmium, and other heavy metal resistance genes were conserved in all wgST31 isolates. By comparing persistent and putative non-persistent isolates, four genes that were all located in the prophage comK might be associated with persistence. These findings enhanced our understanding of the underlying mechanisms of contamination and assist in formulating targeted strategies for the prevention and control of L. monocytogenes transmission from the food processing chain to humans.IMPORTANCE Contamination of food by Listeria monocytogenes at retail level leads to potential consumption of contaminated food with high risk of human infection. Our previous study found persistent contamination of CC87 L. monocytogenes from a retail market in China through pulsed-field gel electrophoresis and multilocus sequence typing. In this study, whole-genome sequencing was used to obtain the highest resolution inference of the source and reasons for persistent contamination; meat grinders and minced meat were the major reservoir of persistent contamination in meat stalls, whereas fishponds were the major reservoir in seafood stalls, with different L. monocytogenes isolates involved. These isolates carried different properties such as plasmids and prophages, which may have contributed to their ability to survive or adapt to the different environments. Our findings suggest that whole-genome sequencing will be an effective surveillance tool to detect persistent L. monocytogenes contamination in retail food markets and to design new control strategies to improve food safety.

Co-occurrence of genes encoding carbapenemase, ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E.coli clinical isolates in Tunisia.

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried blaNDM-1 (n = 11), blaOXA-48 (n = 11), blaNDM-1 + blaOXA-48 (n = 1), blaNDM-1 + blaVIM-1 (n = 1), blaOXA-204 (n = 1), along with blaCTX-M , blaOXA , blaTEM , blaCMY , blaDHA and blaSHV genes variants on conjugative plasmid of IncL/M, IncR, IncFIIK , IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.

Staphylococcus aureus Causing Skin and Soft Tissue Infections in Companion Animals: Antimicrobial Resistance Profiles and Clonal Lineages.

Staphylococcus aureus is a relevant agent of skin and soft tissue infections (SSTIs) in animals. Fifty-five S. aureus comprising all SSTI-related isolates in companion animals, collected between 1999 and 2018 (Lab 1) or 2017 and 2018 (Lab 2), were characterized regarding susceptibility to antibiotics and heavy metals and carriage of antimicrobial resistance determinants. Clonal lineages were established by PFGE, MLST and agr typing. Over half of the isolates (56.4%, 31/55) were methicillin-resistant S. aureus (MRSA), and 14.5% showed a multidrug resistance (MDR) phenotype. Resistance was most frequently observed for beta-lactams (81.8%, related to blaZ and/or mecA), fluoroquinolones (56.4%) and macrolides/lincosamides (14.5%, related to erm(A) or erm(C)). The distributions of heavy-metal MICs allowed the detection of non-wild-type populations associated with several resistance genes. The collection showed genetic diversity, with prevalence of clonal lineage ST22-agrI (45.5%, 25/55), comprising only MRSA isolates, and several less frequently detected clones, including ST5-agrII (14.6%, 8/55), ST398-agrI (9.1%, 5/55) and ST72-agrI (7.3%, 4/55). This work highlights the high frequency of SSTI-related MRSA strains that reflect the clonal lineages circulating both in companion animals and humans in Portugal, reinforcing the need for a One Health approach when studying staphylococci causing infections in companion animals.

High Genetic Diversity and Antimicrobial Resistance in Escherichia coli Highlight Arapaima gigas (Pisces: Arapaimidae) as a Reservoir of Quinolone-Resistant Strains in Brazilian Amazon Rivers.

The increasing prevalence of multi-drug resistant (MDR) Escherichia coli in distinct ecological niches, comprising water sources and food-producing animals, such as fish species, has been widely reported. In the present study, quinolone-resistant E. coli isolates from Arapirama gigas, a major fish species in the Brazilian Amazon rivers and fish farms, were characterized regarding their antimicrobial susceptibility, virulence, and genetic diversity. A total of forty (40) specimens of A. gigas, including 20 farmed and 20 wild fish, were included. Thirty-four quinolone-resistant E. coli isolates were phenotypically tested by broth microdilution, while resistance and virulence genes were detected by PCR. Molecular epidemiology and genetic relatedness were analyzed by MLST and PFGE typing. The majority of isolates were classified as MDR and detected harboring blaCTX-M, qnrA and qnrB genes. Enterotoxigenic E. coli pathotype (ETEC) isolates were presented in low prevalence among farmed animals. MLST and PFGE genotyping revealed a wide genetic background, including the detection of internationally spread clones. The obtained data point out A. gigas as a reservoir in Brazilian Amazon aquatic ecosystems and warns of the interference of AMR strains in wildlife and environmental matrices.

Comparative Analysis of Short- and Long-Read Sequencing of Vancomycin-Resistant Enterococci for Application to Molecular Epidemiology.

Vancomycin-resistant enterococci (VRE) are nosocomial pathogens with genetic plasticity and widespread antimicrobial resistance (AMR). To prevent the spread of VRE in the hospital setting, molecular epidemiological approaches such as pulsed-field gel electrophoresis and multilocus sequence typing have been implemented for pathogen outbreak surveillance. However, due to the insufficient discriminatory power of these methods, whole-genome sequencing (WGS), which enables high-resolution analysis of entire genomic sequences, is being used increasingly. Herein, we performed WGS of VRE using both short-read next-generation sequencing (SR-NGS) and long-read next-generation sequencing (LR-NGS). Since standardized workflows and pipelines for WGS-based bacterial epidemiology are lacking, we established three-step pipelines for SR- and LR-NGS, as a standardized WGS-based approach for strain typing and AMR profiling. For strain typing, we analyzed single-nucleotide polymorphisms (SNPs) of VRE isolates and constructed SNP-based maximum-likelihood phylogenies. The phylogenetic trees constructed using short and long reads showed good correspondence. Still, SR-NGS exhibited higher sensitivity for detecting nucleotide substitutions of bacterial sequences. During AMR profiling, we examined AMR genes and resistance-conferring mutations. We also assessed the concordance between genotypic and phenotypic resistance, which was generally better for LR-NGS than SR-NGS. Further validation of our pipelines based on outbreak cases is necessary to ensure the overall performance of pipelines.

Surveillance Study of Klebsiella pneumoniae in the Giant Panda Revealed High Genetic Diversity and Antibiotic Therapy Challenge.

Klebsiella pneumoniae is not only a worldwide human pathogen, it also effects wildlife, such as the giant panda (Ailuropoda melanoleuca), in which it has recently been evidenced to result in diarrhea, organ failure, and even death. A K. pneumoniae investigation was carried out at the Chengdu Research Base of Giant Panda Breeding in 2018. As part of the investigation, the pulsed-field gel electrophoresis (PFGE) typing, multilocus-sequence typing (MLST), antibiotic resistance profiles (ARPs), and antibiotic resistance genes (ARGs) were studied based on all isolates. Fecal samples were collected from 72 A. melanoleuca from May to December 2018, and a total of 90 K. pneumoniae were isolated from 153 fecal samples. The genotyping results showed that the isolates had high diversity, of which 84 clusters were obtained by PFGE and 57 STs by MLST. The overall trend of the similarity of isolates was the first sample period > second sample period > third sample period, which showed the increasement of genome variability of K. pneumoniae. In addition, 90 isolates showed high resistance to ampicillin, rifampicin, and compound sulfamethoxazole. Of the obtained isolates, 50% carried 6~8 ARPs, and the carrying volume increased during three sample periods, in which we found two isolates carrying 12 and 13 ARPs during the third sample period, respectively. Moreover, a total of 65 ARGs were detected (90.28%, 65/72) in 90 K. pneumoniae samples. Almost all bacteria sampled contained 17 ARGs that belonged to the β-lactamase, Multidrug, MGEs, Aminoglycoside, and Tetracycline, which may be the basis of ARPs of K. pneumoniae. Moreover, the types of Multidrug and MGEs had a greater impact on antibiotic susceptivity of K. pneumoniae. Our results showed that K. pneumoniae has a serious risk of transmission in A. melanoleuca and K. pneumoniae had a high possibility of genome diversity and the risk of drugs tolerance under the large antibiotic usage.

Predominance of community-associated, methicillin-susceptible Staphylococcus aureus infections among hospitalized children and adolescents.

Introduction. Staphylococcus aureus infections cause significant morbidity and mortality in children and adolescents.Gap Statement. There is limited data on the characteristics of S. aureus infections requiring hospitalization in childhood.Aim.To investigate the molecular epidemiology and antibiotic resistance of S. aureus clinical isolates from children and adolescents.Methodology.All S. aureus isolates recovered from patients aged <18 years, admitted to a referral hospital, with culture-proven invasive or non-invasive infections during the 4 year period 2015 to 2018 were analysed for antimicrobial resistance, virulence genes, PFGE and multilocus sequence typing (MLST). Cases were assigned to community-associated, community-onset healthcare-associated or hospital-associated infections based on epidemiological case definitions.Results.Among 139 S. aureus infections, 88.5 % (123/139) were caused by methicillin-susceptible isolates (MSSA) and 73.4 % (102/139) were classified as community-associated infections. tst and lukS/lukF-PV genes were more common among MRSA as compared to MSSA isolates (tst, p 0.04; lukS/lukF-PV, p 0.007). Invasive disease was noted in 22/139 patients (15.8 %). Staphylococcal scalded skin syndrome caused by fusidic-resistant MSSA increased over time (22.8 % in 2017-2018 vs 8.3 % in 2015-2016, OR 3.24; 95 % CI 1.10-8.36; P 0.03). By PFGE genotyping, 22 pulsotypes were identified. A total of five sequence types (STs) were identified among 58 isolates analysed by MLST. More than one third of MSSA isolates (40/123, 32.5 %) and 13/23 (56.5 %) of SSSS isolates belonged to pulsotype 1, classified as sequence type 121 (ST121). MRSA isolates were equally distributed to pulsotypes A (ST30), B (ST239), C (ST80), H (ST225). ST121 isolates carried fnbA (40/40), eta/etb genes (29/40), exhibited high resistance to fusidic acid and were increasingly resistant to mupirocin.Conclusion.In our population, community-associated MSSA was the predominant cause of S. aureus infections characterized by polyclonality, increasing resistance to fusidic acid and mupirocin. PFGE type 1 ST121 clone, harboured exfoliative toxin genes and was associated with rising trends of SSSS.

Molecular Characterization of Staphylococcus aureus Strains Isolated from Mobile Phones.

The widespread use of mobile phones (MP) among healthcare personnel might be considered as an important source of contamination. One of the most pathogenic bacteria to humans is Staphylococcus aureus, which can be transmitted through the constant use of MP. Nevertheless, which specific type of strains are transmitted and which are their sources have not been sufficiently studied. The aim of this study is to determine the source of contamination of MP and characterize the corresponding genotypic and phenotypic properties of the strains found. Nose, pharynx, and MP samples were taken from a group of health science students. We were able to determinate the clonality of the isolated strains by pulsed-field gel electrophoresis (PFGE) and spa gene typing (spa-type). Adhesin and toxin genes were detected, and the capacity of biofilm formation was determined. Several of the MP exhibited strains of S. aureus present in the nose and/or pharynx of their owners. methicillin-susceptible Staphylococcus aureus (MSSA), hospital-acquired methicillin-resistant S. aureus (HA-MRSA), and community-acquired methicillin-resistant S. aureus (CA-MRSA) strains were found, which indicated a variety of genotypes. This study concludes that MP can be contaminated with the strains of S. aureus present in the nose and/or pharynx of the owners; these strains can be of different types and there is no dominant genotype.

Longitudinal Monitoring Reveals Persistence of Colistin-Resistant Escherichia coli on a Pig Farm Following Cessation of Colistin Use.

Colistin-resistant bacteria harboring plasmid-mediated mcr genes are of concern as they may be a cause of serious nosocomial infections. It is hypothesized that cessation of colistin use as a feed additive for pigs will reduce the occurrence and distribution of mcr genes in farms. The aim of this study was to investigate this hypothesis by longitudinal monitoring and characterizing of mcr positive Escherichia coli (MCRPE) isolates after colistin was withdrawn on a central Thailand pig farm that previously had a high frequency of MCRPE. Colistin use ceased at the beginning of 2017, and subsequently 170 samples were collected from farrowing sows and suckling piglets (n = 70), wastewater (n = 50) and farm workers (n = 50) over a 3.5-year period. Bacteria were identified by MALDI-TOF mass spectrometry and minimal inhibitory concentrations were determined by broth microdilution. The antibiogram of mcr positive E. coli isolates was determined using the Vitek2 automated susceptibility machine, and multiplex and simplex PCRs were performed for mcr-1–8 genes. MCRPE containing either mcr-1 or mcr-3 were isolated from pigs throughout the investigation period, but with a declining trend, whereas MCRPE isolates were recovered from humans only in 2017. MCRPE were still being recovered from wastewater in 2020. Most MCRPE isolates possessed the virulence genes Stap, Stb, or Stx2e, reflecting pathogenic potential in pigs, and showed high rates of resistance to ampicillin, gentamicin and tetracycline. Pulsed-field gel electrophoresis and multi-locus sequence typing showed that diverse MCRPE clones were distributed on the farm. The study identified a decline of pathogenic MCRPE following withdrawal of colistin, with pigs being the primary source, followed by wastewater. However, short-term therapeutic usage of other antibiotics could enhance the re-occurrence of mcr-carrying bacteria. Factors including the environment, management, and gene adaptations that allow maintenance of colistin resistance require further investigation, and longer-term studies are needed.

A Longitudinal Nine-Year Study of the Molecular Epidemiology of Carbapenemase-Producing Enterobacterales Isolated From a Regional Hospital in Taiwan: Predominance of Carbapenemase KPC-2 and OXA-48.

Enterobacterales clinical isolates are now being resistant to clinically achievable concentrations of most commonly used antibiotics that makes treatment of hospitalized patients very challenging. We hereby determine the molecular characteristics of carbapenemase genes in carbapenem-resistant Enterobacterales (CRE) isolates in Taiwan. A total of 455 CRE isolates were identified between August 2011 to July 2020. Minimum inhibitory concentrations for selected carbapenems were tested using Vitek 2, and carbapenemase genes were determined using polymerase chain reaction in combination with sequencing. Phenotypic detection of carbapenemase was determined by modified carbapenem inactivation method (mCIM) and EDTA-modified carbapenem inactivation method (eCIM) to validate our PCR screening results. Pulsed-field gel electrophoresis (PFGE) was used to determine the clonality of carbapenemase-producing Enterobacterales (CPE) isolates, and the transferability of carbapenemase-carrying plasmids was determined by conjugation assays. A slight increase in carbapenem-resistant E. coli (CREC) was observed, however, the prevalence of carbapenem-resistant K. pneumoniae (CRKP) was steady, during 2011–2020. The dominant species among our CRE was K. pneumoniae (270/455, 59.3%), followed by E. coli (81/455, 17.8%), Morganella morganii (32/455, 7.0%), and Enterobacter cloacae (25/455, 5.5%). From 2011 to 2020, the total percentage of CPE increased steadily, accounting for 61.0% of CRE in 2020. Moreover, 122 of 455 CRE isolates (26.8%) were CPE. Among the CPE isolates, the dominant carbapenemase gene was blaOXA–48–like (54/122, 44.3%), and the second most common carbapenemase gene was blaKPC–2 (47/122, 38.5%). The sensitivity and specificity for mCIM to detect carbapenemase in the 455 isolates were both 100% in this study. The PFGE results showed that 39 carbapenemase-producing E. coli and 69 carbapenemase-producing K. pneumoniae isolates carrying blaKPC–2 and/or blaNDM–5 could be classified into 5 and 12 clusters, respectively. In conclusion, our results showed an increase in CPE isolates in Taiwan. Moreover, the distribution of carbapenemase and antimicrobial susceptibility in CPE were associated with PFGE typing.