Pulsed-field Gel Electrophoresis Research Articles

Epidemiological Characteristics of Shiga Toxin-Producing Escherichia coli Responsible for Infections in the Polish Pediatric Population.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum β-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.

Phenotypic and molecular characterisation of Salmonella spp. isolates in healthy poultry

ABSTRACT 1. Epidemiological surveillance of Salmonella spp. serves as a primary tool for maintaining the health of poultry flocks. Characterising circulating serotypes is crucial for implementing control and prevention measures. This study conducted phenotypic and molecular characterisation of S. enterica Pullorum, S. enterica Heidelberg, and S. enterica Corvalis isolated from broiler chickens during slaughtering. 2. All strains were susceptible to gentamicin, neomycin and norfloxacin. However, resistance rates exceeded 50% for ciprofloxacin and tiamulin, irrespective of the serotype. Approximately 64% of strains were classified as multidrug-resistant, with S. enterica Heidelberg strains exhibiting significantly higher overall resistance. The isolates demonstrated the ability to adhere and produce biofilm at a minimum of three temperatures, with S. enterica Pullorum capable of biofilm production at all temperatures encountered during poultry rearing. 3. Each strain possessed between two and seven different virulence-associated genes. Genetic similarity, as indicated by pulsed field gel electrophoresis, exceeded 90% for all three serotypes and strains were classified in the R5 ribotype by PCR, regardless of serotype. Sequencing revealed high similarity among all strains, with homology ranging from 99.61 to 100% and all were classified to a single cluster. 4. The results suggested a clonal relationship among the strains, indicating the possible circulation of a unique clonal group of S. enterica Pullorum in the southern region of Brazil.

Hospital water environment and antibiotic use: Key factors in a nosocomial outbreak of carbapenemase-producing Serratia marcescens

BackgroundThe healthcare water environment is a potential reservoir of carbapenem-resistant organisms (CROs). Here, we report the role of the water environment as a reservoir and the infection control measures applied to suppress a prolonged outbreak of Klebsiella pneumoniae carbapenemase-producing Serratia marcescens (KPC-SM) in two intensive care units (ICUs). MethodsThe outbreak occurred in the ICUs of a tertiary hospital from October 2020 to July 2021. Comprehensive patient contact tracing and environmental assessments were conducted, and a case–control study was performed to identify factors associated with the acquisition of KPC-SM. Associations among isolates were assessed via pulsed-field gel electrophoresis (PFGE). Antibiotic usage was analyzed. . ResultsThe outbreak consisted of two waves involving a total of 30 patients with KPC-SM. Multiple environmental cultures identified KPC-SM in a sink, a dirty utility room, and a communal bathroom shared by the ICUs, together with the waste bucket of a continuous renal replacement therapy (CRRT) system. The genetic similarity of the KPC-SM isolates from patients and the environment was confirmed by PFGE. A retrospective review of 30 cases identified that the use of CRRT and antibiotics were associated with acquisition of KPC-SM (p < 0.05). There was a continuous increase in the use of carbapenems; notably, the use of colistin has increased since 2019. ConclusionOur study demonstrates that CRRT systems, along with other hospital water environments, are significant potential sources of resistant microorganisms, underscoring the necessity of enhancing infection control practices in these areas.

Residual risk of Pseudomonas aeruginosa waterborne contamination in intensive care unit despite the presence of filters on all water points-of-use

To assess the residual risk of waterborne contamination by Pseudomonas aeruginosa (PA) from water network colonized by a single genotype (Sequence Type (ST)299) despite the presence of antimicrobial filters in a medical intensive care unit (ICU). During the first 19-month period after ICU opening, water network contamination was assessed monthly by collecting water upstream the filters. Downstream water was also sampled to assess filters efficiency. PA clinical isolates from patients were collected and compared to the waterborne ST299-PA by multiplex-rep PCR, Pulsed-Field Gel Electrophoresis (PFGE) and whole-genome sequencing. Cross-transmission events occurring independently of the genotype of PA involved were also assessed. From 449 samples of filtered water, 1.3% were positive for PA in inoculum varying between 1 to 104 Colony Forming Unit (CFU)/100mL according to the tap. All PA hydric isolates belonged to PA-ST299 and displayed <2 Single Nucleotide Polymorphisms (SNPs). Among 278 clinical isolates from 122 patients, 10 isolates in 5 patients showed identical profiles to the hydric PA-ST299 clone in both multiplex-rep PCR and PFGE and differed by <5 SNPs on average, confirming the water network reservoir as the source of contamination by PA for 4.09% of patients. Cross-transmission events by other genotypes of PA than PA-ST299 were responsible for the contamination of 1.75 % of patients. Antimicrobial filters are not sufficient to preserve patients from waterborne pathogens when the water network is highly contaminated. The microbiological survey of filtered water may be needed in units hosting at-risk patients for PA infections, even when all water points-of-use are protected by filters.

Best practice guidelines on genetic diagnostics of facioscapulohumeral muscular dystrophy: Update of the 2012 guidelines.

The gold standard for facioscapulohumeral muscular dystrophy (FSHD) genetic diagnostic procedures was published in 2012. With the increasing complexity of the genetics of FSHD1 and 2, the increase of genetic testing centers, and the start of clinical trials for FSHD, it is crucial to provide an update on our knowledge of the genetic features of the FSHD loci and renew the international consensus on the molecular testing recommendations. To this end, members of the FSHD European Trial Network summarized the evidence presented during the 2022 ENMC meeting on Genetic diagnosis, clinical outcome measures, and biomarkers. The working group additionally invited genetic and clinical experts from the USA, India, Japan, Australia, South-Africa, and Brazil to provide a global perspective. Six virtual meetings were organized to reach consensus on the minimal requirements for genetic confirmation of FSHD1 and FSHD2. Here, we present the clinical and genetic features of FSHD, specific features of FSHD1 and FSHD2, pros and cons of established and new technologies (Southern blot in combination with either linear or pulsed-field gel electrophoresis, molecular combing, optical genome mapping, FSHD2 methylation analysis and FSHD2 genotyping), the possibilities and challenges of prenatal testing, including pre-implantation genetic testing, and the minimal requirements and recommendations for genetic confirmation of FSHD1 and FSHD2. This consensus is expected to contribute to current clinical management and trial-readiness for FSHD.

[Comparison of Three Different Methods in Investigating the Molecular Epidemiology of Carbapenem-Resistant Acinetobacter baumannii Clinical Isolates].

The aim of the study was to evaluate the relationship between carbapenem-resistant Acinetobacter baumannii isolates carrying oxacillinase-type carbapenemase genes with "international high-risk clones" (IC I, II, and III) by different molecular epidemiological methods and to statistically compare the concordance and discrimination power of the methods. Carbapenem-resistant and moderately susceptible A.baumannii isolates from non-repeating blood cultures of 72 patients were included in the study. The presence of "blaOXA-23 , blaOXA-24 , blaOXA-51 ve blaOXA-58 " genes within OXA-type carbapenemases was detected by polymerase chain reaction (PCR) method and confirmed by DNA sequence analysis. Pulsed f ield gel electrophoresis (PFGE), multilocus sequence typing (MLST) and matrix-assisted laser desorption/ ionization time- of-flight mass spectrometry (MALDI-TOF MS) analyses were performed to evaluate the clonal relations of IC I, II and III clones together with clinical isolates. In the statistical comparison of the methods, discrimination power was evaluated by Simpson index of diversity (SID) and concordance by "Wallace coefficient". All of the isolates were found to carry blaOXA-23 and blaOXA-51 genes. As a result of the bioinformatic analysis of the four isolates selected for sequence analysis; blaOXA-23 and blaOXA-51 genes were detected in the selected isolates, and the analysis of two isolates carrying blaOXA-51 gene showed 99% similarity with blaOXA-92 gene. The isolates were clustered into five pulsotypes (A, B, C, D and E) according to ≥ 85% similarity coefficient by PFGE. The isolates and RUH 875, RUH 134, LUH 5875 strains belonging to high-risk clones ICI, ICII and ICIII, respectively, were divided into five main groups [A (n= 58), B (n= 8), C (n= 4), D (n= 4) and E (n= 1)] and 10 subgroups (A1, A2, A4, A5, A6, A9, B1, B4, C3, D1) by PFGE. IC clone III (E1) and seven strains showed singleton PFGE profiles (A3, A7, A8, B2, B3, C1, C2). ICII was found in A5 subtype, ICI in C1 subtype and ICIII in E1 subtype. By PFGE subtype groups, 18 pulsotypes were determined and ST1, ST2, ST81, ST157 and ST604 sequence types were found in 20 isolates randomly selected from pulsotypes according to MLST Pasteur scheme (cpn60, fusA, gltA, pyrG, recA, rplB, rpoB). Principal component analysis (PCA) of the spectra of 72 A. baumannii isolates and ICI, ICII and ICIII clones was performed by MALDI-TOF MS. In PCA analysis, the cluster distance level was defined as 1.5 and the isolates were divided into three clusters. IC clone I, II and III together with 70 clinical isolates were grouped in one cluster, while two clinical isolates (AB083 and AB0115) formed singleton clusters. There was no significant agreement between MALDI-TOF MS; MLST and PFGE data according to Wallace coefficient. It was found that PFGE method gave significant results in terms of discrimination power with SID coefficient, MALDI-TOF MS PCA analysis had the lowest discrimination power value, and the Wallace coefficient result of PFGE and MLST was concordant. In conclusion, MALDI-TOF MS may not function as a gold standard method like PFGE and MLST for epidemiological analysis in A.baumannii species and the epidemiological typing protocols used for MALDI-TOF MS need to be improved and developed.

In vitro activity of cefiderocol against carbapenem-resistant Acinetobacter baumannii carrying various β-lactamase encoding genes.

This study aimed to determine the in vitro efficacy of cefiderocol in carbapenem-resistant Acinetobacter baumannii (CRAB) isolates and evaluate the disk-diffusion (DD) method as an alternative method to broth-microdilution (BMD). Totally 89 CRAB isolates were included. Cluster analysis was determined by Pulsed-Field Gel Electrophoresis (PFGE). Resistance genes; blaOXA-51, blaOXA-23, blaOXA-24, blaOXA-58,blaPER-1, blaNDM, blaIMP and mcr-1 were screened. Cefiderocol susceptibility testing was performed by both DD and BMD. Interpretation was made according to EUCAST and CLSI. Categorical agreement (CA), minor errors (mEs), major errors (MEs), and very major errors (VMEs) were determined. PFGE revealed 5 distinct pulsotypes; 86 of the isolates were extensively drug-resistant (XDR). All the isolates were negative for blaNDM, blaIMP, mcr-1, while positive for blaOXA-58 and blaOXA51. blaPER-1 was positive for 33.7%; blaOXA-23 for 74.2%; blaOXA-24 for 12.3%. According to CLSI, the MEs rate was 1.85%, mEs was 7.86% and there were no VMEs. According to EUCAST, MEs rate was 3.70%, there were no mEs and VMEs. CA was 91% for CLSI and 97.8% for EUCAST. MICs of cefiderocol against A. baumannii isolates ranged from 0.06 to > 128mg/L, with MIC50 and MIC90 values of 0.5 and > 128mg/L, respectively. Cefiderocol susceptibility was 60.7% in CRAB isolates. MIC50, MIC90 of blaPER-1 positive and blaPER-1 negative groups were > 128/>128 and 0.25/>128mg/L. A correlation between the presence of blaPER-1 and cefiderocol resistance was observed (p < 0.0001). Among colistin-resistant isolates, the presence of blaPER-1 was 47.1% and 75% of them were resistant to cefiderocol respectively.

The airway microbiome of persons with cystic fibrosis correlates with acquisition and microbiological outcomes of incident Stenotrophomonas maltophilia infection.

Chronic infection with Stenotrophomonas maltophilia in persons with cystic fibrosis (pwCF) has been linked to an increased risk of pulmonary exacerbations and lung function decline. We sought to establish whether baseline sputum microbiome associates with risk of S. maltophilia incident infection and persistence in pwCF. pwCF experiencing incident S. maltophilia infections attending the Calgary Adult CF Clinic from 2010-2018 were compared with S. maltophilia-negative sex, age (+/-2 years), and birth-cohort-matched controls. Infection outcomes were classified as persistent (when the pathogen was recovered in ≥50% of cultures in the subsequent year) or transient. We assessed microbial communities from prospectively biobanked sputum using V3-V4 16S ribosomal RNA (rRNA) gene sequencing, in the year preceding (Pre) (n = 57), at (At) (n = 22), and after (Post) (n = 31) incident infection. We verified relative abundance data using S. maltophilia-specific qPCR and 16S rRNA-targeted qPCR to assess bioburden. Strains were typed using pulse-field gel electrophoresis. Twenty-five pwCF with incident S. maltophilia (56% female, median 29 years, median FEV1 61%) with 33 total episodes were compared with 56 uninfected pwCF controls. Demographics and clinical characteristics were similar between cohorts. Among those with incident S. maltophilia infection, sputum communities did not cluster based on infection timeline (Pre, At, Post). Communities differed between the infection cohort and controls (n = 56) based on Shannon Diversity Index (SDI, p = 0.04) and clustered based on Aitchison distance (PERMANOVA, p = 0.01) prior to infection. At the time of incident S. maltophilia isolation, communities did not differ in SDI but clustered based on Aitchison distance (PERMANOVA, p = 0.03) in those that ultimately developed persistent infection versus those that were transient. S. maltophilia abundance within sputum was increased in samples from patients (Pre) relative to controls, measuring both relative (p = 0.004) and absolute (p = 0.001). Furthermore, S. maltophilia abundance was increased in sputum at incident infection in those who ultimately developed persistent infection relative to those with transient infection, measured relatively (p = 0.04) or absolute (p = 0.04), respectively. Microbial community composition of CF sputum associates with S. maltophilia infection acquisition as well as infection outcome. Our study suggests sputum microbiome may serve as a surrogate for identifying infection risk and persistence risk.

Usefulness and Limitations of PFGE Diagnosis and Nucleotide Sequencing Method in the Analysis of Food Poisoning Pathogens Found in Cooking Employees.

In the case of a food poisoning outbreak, it is essential to understand the relationship between cooking workers and food poisoning. Many biological diagnostic methods have recently been developed to detect food poisoning pathogens. Among these diagnostic tools, this study presents PCR-based pulsed-field gel electrophoresis and nucleotide sequencing diagnostic analysis results for diagnosing food poisoning outbreaks associated with cooking employees in Chungcheongnam-do, Republic of Korea. Pulsed-field gel electrophoresis was useful in identifying the food poisoning outbreaks caused by Staphylococcus aureus and Enteropathogenic Escherichia coli. In the case of Norovirus, nucleotide sequencing was used to identify the relationship between cooking workers and the food poisoning outbreak. However, it is difficult to determine whether cooking employees directly caused the food poisoning outbreaks based on these molecular biological diagnostic results alone. A system is needed to integrate epidemiological and diagnostic information to identify a direct correlation between the food poisoning outbreak and cooking employees.

Characteristics of the Genetic Spread of Carbapenem-Resistant Acinetobacter baumannii in a Tertiary Greek Hospital.

Acinetobacter baumannii (Ab) has increasingly been identified as a cause of hospital-acquired infections and epidemics. The rise of carbapenem-resistant Acinetobacter baumannii (CRAB) poses significant challenges in treatment. Nosocomial outbreaks linked to CRAΒ A. baumannii strains have been reported worldwide, including in Greece. This study aimed to analyze the molecular epidemiology trends of multidrug-resistant A. baumannii isolates in a tertiary hospital in Athens, Greece. A total of 43 clinical isolates of extensively drug-resistant (XDRAB), pan-drug-resistant (PDRAB), and CRAB were collected from patients suffering from blood infection, hospitalized between 2016 and 2020 at the internal medicine clinics and the ICU. A.baumannii isolates underwent testing for Ambler class B and D carbapenemases and the detection of ISAba1, and were typed, initially, using pulsed-field gel electrophoresis, and, subsequently, using sequence-based typing and multiplex PCR to determine European Clone lineages. The blaOXA-23 gene accompanied by ISAba1 was prevalent in nearly all A. baumannii isolates, except for one carrying blaOXA-58. The intrinsic blaOXA-51-like gene was found in all isolates. No Ambler class B carbapenemases (VIM, NDM) were detected. Isolates were grouped into four PF-clusters and no one-cluster spread was documented, consistent with the absence of outbreak. The study indicated that XDR/PDR-CRAB isolates predominantly produce OXA-23 carbapenemase and belong to European Clone II. Further research is needed to understand the distribution of resistant bacteria and develop effective prevention and control strategies.

Pathogenic characteristics of an aggregated diarrhea event caused by Plesiomonas shigelloides from stream.

This study aimed to investigate the cause of a foodborne disease outbreak in Huzhou on August 14, 2023. Multiple enteropathogens were detected using FilmArray, and the pathogen was subsequently isolated and cultured from anal swabs of the cases and stream water. The isolated strains were identified using VITEK MS, and antimicrobial susceptibility test, pulsed field gel electrophoresis (PFGE) molecular typing, and whole genome sequencing (WGS) were performed on the isolates of Plesiomonas shigelloides. Gene annotation and sequence alignment were used to analyze the virulence genes and drug resistance genes of the strains. A phylogenetic tree was constructed based on single nucleotide polymorphism (SNP), and homology analysis was conducted to trace the origin of P. shigelloides. A total of 7 strains of P.shigelloides were isolated, with 3 from stream water and 4 from anal swabs. All 7 strains exhibited the same PFGE pattern and showed resistance to amikacin, trimethoprim-sulfamethoxazole, chloramphenicol, tetracycline, cefazolin, streptomycin, and florfenicol. The isolated strains carried the same resistance genes and virulence factors. In the sequences of the isolated strains from this outbreak, 11 mutation sites were detected. The phylogenetic tree based on SNP sites showed that these strains were homologous. This foodborne disease outbreak caused by P.shigelloides was the first reported in Huzhou. WGS can be used as a complementary method to PFGE for epidemiological investigations of disease outbreaks.

Persistence of viable but nonculturable Legionella pneumophila state in hospital water systems: A hidden enemy?

There is little evidence of the long-term consequences of maintaining sanitary hot water at high temperatures on the persistence of Legionella in the plumbing system. The aims of this study were to describe the persistence and genotypic variability of L. pneumophila in a hospital building with two entirely independent hot water distribution systems, and to estimate the thermotolerance of the genotypic variants by studying the quantity of VBNC L. pneumophila. Eighty isolates from 55 water samples obtained between the years 2012–2017 were analyzed. All isolates correspond to L. pneumophila serogroup 6. The isolates were discriminated in four restriction patterns by pulsed-field gel electrophoresis. In one installation, pattern A + Aa predominated, accounting for 75.8 % of samples, while the other installation exhibited pattern B as the most frequent (81.8 % of samples; p < 0.001). The mean temperature of the isolates was: 52.6 °C (pattern A + Aa) and 55.0 °C (pattern B), being significantly different. Nine strains were selected as representative among patterns to study their thermotolerance by flow-cytometry after 24 h of thermic treatment. VBNC bacteria were detected in all samples. After thermic treatment at 50 °C, 52.0 % of bacteria had an intact membrane, and after 55 °C this percentage decreased to 23.1 %. Each pattern exhibited varying levels of thermotolerance. These findings indicate that the same hospital building can be colonized with different predominant types of Legionella if it has independent hot water installations. Maintaining a minimum temperature of 50 °C at distal points of the system would allow the survival of replicative L. pneumophila. However, the presence of Legionella in hospital water networks is underestimated if culture is considered as the standard method for Legionella detection, because VBNC do not grow on culture plates. This phenomenon can carry implications for the Legionella risk management plans in hospitals that adjust their control measures based on the microbiological surveillance of water.

Antibiotic-Resistance Profiles and Genetic Diversity of Shigella Isolates in China: Implications for Control Strategies.

The urgent need for comprehensive and systematic analyses of Shigella as the key pathogen led us to meticulously explore the epidemiology and molecular attributes of Shigella isolates. Accordingly, we procured 24 isolates (10 from Xinjiang and 14 from Wuhan, China) and performed serotype identification and antimicrobial susceptibility testing. Resistance gene detection and homology analysis by polymerase chain reaction and pulsed-field gel electrophoresis (PFGE), respectively, were performed for genetic diversity analysis. All isolates were identified as Shigella flexneri, with 70% (35.4-91.9%) and 30% (8.1-64.6%) of the Xinjiang isolates and 85.7% (56.2-97.5%) and 14.3% (2/14, 2.5-43.9%) of the Wuhan isolates belonging to serotype 2a and serotype 2b, respectively. All isolates displayed resistance to at least two antibiotics and complete resistance to ampicillin. Multidrug resistance (MDR) was recorded in 70.8% (48.8-86.6%) of isolates, with Xinjiang isolates exhibiting relatively higher resistance to ampicillin-sulbactam, piperacillin, ceftriaxone, and aztreonam. Conversely, Wuhan isolates displayed higher MDR and resistance to tetracycline, ciprofloxacin, levofloxacin, and cefepime relative to Xinjiang isolates. Molecular scrutiny of antibiotic-resistance determinants revealed that blaTEM was the main mechanism of ampicillin resistance, blaCTX-M was the main gene for resistance to third- and fourth-generation cephalosporins, and tetB was the predominant gene associated with tetracycline resistance. Four Xinjiang and seven Wuhan isolates shared T1-clone types (>85%), and two Xinjiang and one Wuhan isolates were derived from the T6 clone with a high similarity of 87%. Six PFGE patterns (T1, T2, T5, T6-3, T8, and T10) of S. flexneri were associated with MDR. Thus, there is a critical need for robust surveillance and control strategies in managing Shigella infections, along with the development of targeted interventions and antimicrobial stewardship programs tailored to the distinct characteristics of Shigella isolates in different regions of China.

Enterotoxigenic Staphylococcus aureus in Brazilian artisanal cheeses: Occurrence, counts, phenotypic and genotypic profiles

The present study aimed to assess the occurrence and counts of Staphylococcus aureus in Brazilian artisanal cheeses (BAC) produced in five regions of Brazil: Coalho and Manteiga (Northeast region); Colonial and Serrano (South); Caipira (Central-West); Marajó (North); and Minas Artisanal cheeses, from Araxá, Campos das Vertentes, Cerrado, Serro and Canastra microregions (Southeast). The resistance to chlorine-based sanitizers, ability to attach to stainless steel surfaces, and antibiogram profile of a large set of S. aureus strains (n = 585) were assessed. Further, a total of 42 isolates were evaluated for the presence of enterotoxigenic genes (sea, seb, sec, sed, see, seg, sei, sej, and ser) and submitted to typing using pulsed-field gel electrophoresis (PFGE). BAC presented high counts of S. aureus (3.4–6.4 log CFU/g), varying from 25 to 62.5%. From the S. aureus strains (n = 585) assessed, 16% could resist 200 ppm of sodium hypochlorite, whereas 87.6% produced strong ability to attach to stainless steel surfaces, corroborating with S. aureus ability to persist and spread in the environment. Furthermore, the relatively high frequency (80.5%) of multidrug-resistant S. aureus and the presence of enterotoxin genes in 92.6% of the strains is of utmost attention. It reveals the lurking threat of SFP that can survive when conditions are favorable. The presence of enterotoxigenic and antimicrobial-resistant strains of S. aureus in cheese constitutes a potential risk to public health. This result calls for better control of cheese contamination sources, and taking hygienic measures is necessary for food safety. More attention should be paid to animal welfare and hygiene practices in some dairy farms during manufacturing to enhance the microbiological quality of traditional cheese products.

Molecular characterization of Escherichia coli isolates recovered from broilers with cellulitis

Avian cellulitis in broilers, caused by avian pathogenic Escherichia coli, is a major cause for carcass rejections during meat inspection, resulting in significant economic losses. In this study, we analysed E. coli isolates obtained from broiler chickens affected by cellulitis for their genetic relatedness and antimicrobial resistance phenotype and genotype. The objective was to determine whether there is a clonal spread or whether these clinical isolates differ. For this purpose, E. coli was isolated from swab samples collected from diseased broilers across 77 poultry farms in Germany, resulting in 107 isolates. These isolates were subjected to serotyping, PCR-based phylotyping and macrorestriction analysis with subsequent pulsed-field gel-electrophoresis for typing purposes. In addition, the presence of virulence genes associated with avian pathogenic E. coli (APEC) was investigated by PCR. Antimicrobial susceptibility of the isolates was examined by the disk diffusion method according to CLSI guidelines and subsequently, the presence of corresponding resistance genes was investigated by PCR. Typing results revealed that a significant proportion of the isolates belonged to serotype O78:K80, which is one of the major APEC serotypes. Phylogenetic grouping showed that phylogenetic group D was most commonly represented (n = 49). Macrorestriction analysis showed overall heterogenous results, however, some clustering of closely related isolates was observed. The level of antimicrobial resistance was high, with 83.8% of isolates non-susceptible to at least one class of antimicrobial agents and 40% of isolates showing resistance to at least three classes. The most frequently observed resistance was to ampicillin, mediated by blaTEM (n = 56). However, few isolates were non-susceptible to ciprofloxacin (n = 8) and none of the isolates was resistant to 3rd generation cephalosporins or carbapenems. Overall, the results show that genetically diverse APEC associated with avian cellulitis can be found among and within German poultry farms. While most isolates were antimicrobial resistant, resistance levels to high(est) priority critically important antimicrobials were low.

High Prevalence of Carbapenem-Resistant Enterobacterales Producing OXA-48 among Carbapenem-Resistant Isolates in a Regional Hospital in Central Taiwan.

In response to the increasing number of carbapenem-resistant Enterobacterales (CRE), we investigated carbapenemase-producing Klebsiella pneumoniae and non-K. pneumoniae epidemiology and genetics. We collected 76 clinical Enterobacterales and 4 stool surveillance Escherichia coli isolates resistant to ertapenem or imipenem. Using polymerase chain reaction (PCR) and DNA sequencing, we assessed carbapenemases, extended-spectrum β-lactamases, and AmpC β-lactamases. Molecular typing via pulsed-field gel electrophoresis (PFGE) and conjugation experiments were conducted to examine resistance gene transfer. Among the 80 isolates, 96.2% harbored at least one carbapenemase gene, with blaOXA-48 in 87.5%. KPC-2 and IMP-8 carbapenemases were found in 15.0 and 22.5% of the isolates, respectively, with 27.5% having 2 or more carbapenemase genes. The PFGE analysis revealed the presence of diverse genotypes. PCR-based plasmid replicon typing identified IncA/C as the most prevalent type among K. pneumoniae isolates (26/29), and IncF and IncFIB among E. coli isolates (22/28). Conjugal transfer was successful for plasmids encoding OXA-48, CTX-M-3, CTX-M-14, CMY-2, and other β-lactamases, except the KPC-2 gene. In conclusion, our study highlights high carbapenemase prevalence in CRE, primarily OXA-48. Multiple carbapenemases within strains were common, and PFGE showed diverse patterns in these carbapenem-resistant isolates.

High prevalence of carbapenem resistance and clonal expansion of blaNDM gene in Klebsiella pneumoniae isolates in an Iranian referral pediatric hospital

BackgroundThe increasing global concern regarding antibiotic resistance necessitates in-depth studies to comprehend the phenotypic and genotypic characteristics of resistant bacterial strains. This study aimed to investigate the prevalence, antibiotic resistance profiles, and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in an Iranian referral pediatrics hospital. Methods: In this study, we examined CRKP isolates collected from hospitalized pediatric patients across various wards. The isolates underwent antimicrobial susceptibility testing, the polymerase chain reaction (PCR) analysis for carbapenemase genes (blaNDM, blaVIM and blaIMP), and genetic relatedness assessment using pulsed-field gel electrophoresis (PFGE).ResultsAmong 166 K. pneumoniae isolates, 54 (32.5%) exhibited resistance to carbapenems. Notably, all these resistant isolates were resistant to imipenem, with 35 (65%) displaying resistance to both imipenem and meropenem. Of the 54 CRKP isolates, 24 (44%) were metallo-β-lactamases (MBL)-producing. The prevalence of the blaNDM gene among CKCP and MBL-producing isolates was 20% (n = 11) and 44% (n = 24), respectively. The blaVIM and blaIMP genes were not detected in any of the isolates. Twenty-six CRKP isolates (48%) were recovered from ICUs. PFGE analysis of CRKP isolates revealed 20 clusters, with cluster S being the most prevalent, comprising 24% of the total (n = 13).ConclusionOur study reveals a concerning prevalence of carbapenem resistance in K. pneumoniae isolates. Specifically, the detection of the blaNDM gene in 20% of CRKP isolates, with a significant proportion (82%) observed in isolated CRKP from the ICUs and emergency departments, underscores the potential clonal expansion of these resistant strains within these critical hospital wards.

Epidemiology and molecular characterization of carbapenem-resistant Klebsiella pneumoniae isolated from neonatal intensive care units in General Hospital of Ningxia Medical University, China, 2017-2021.

This study aimed to retrospectively investigate the epidemiology and molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates from neonatal intensive care units (NICU) between 2017 and 2021. The antibacterial susceptibility of all strains was assessed using the VITEK 2 compact system. The presence of antibiotic resistance, virulence genes, sequence types (STs), capsular (K) types, and the wzi genes was determined through polymerase chain reaction (PCR). Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI. Additionally, the virulence potential of peg344-positive strains was evaluated using the string test and mouse intraperitoneal infection models. Whole-genome sequencing was conducted on the DNB system and PacBio platforms. A total of 46 CRKP isolates were collected during the study period. Out of these, 93.47% (43/46) were identified as CRKP strains belonging to the ST76-K10 type carrying blaNDM-5. It was observed that CRKP infection resulted in more severe clinical symptoms compared to CRKP colonization. Among the CRKP strains, a hypervirulent CRKP strain called KP-63, belonging to the ST23 type, was identified. This strain exhibited high mortality in the mouse infection model and was found to possess virulence genes. Genomic alignment analysis revealed a significant similarity between the virulence plasmid from KP-63 strain (pKP-63) and pK2044 from the hypervirulent K. pneumoniae strain NTUH-2044. There has been a potential dissemination of ST76-K10 type CRKP carrying blaNDM-5 in the NICU at Ningxia Hospital. Neonatal CRKP infection has been found to cause more severe clinical symptoms than colonization. Furthermore, we have discovered a CR-hvKP strain of ST23 with serotype K1, which exhibits a significant resemblance in its virulent plasmid to pK2044. Therefore, it is crucial to enforce effective measures to restrict the spread and hinder the evolution of CRKP within the hospital.

Virulence properties of Listeria monocytogenes isolated from meat and meat contact surfaces in a slaughterhouse.

Listeria monocytogenes is a ubiquitous microorganism that is isolated from a variety of sources such as soil, water, decaying vegetation, sewage, animal feeds, silage, farm environments and food-processing environments. This study aimed to determine the prevalence, serogroups, biofilm formation, virulence factor genes, and genetic relationships of L. monocytogenes strains isolated from beef meat and meat contact surfaces obtained from a slaughterhouse in Burdur, Turkey. In this study, a total of 179 beef meat and meat contact surface samples were analyzed for the presence of L. monocytogenes by polymerase chain reaction (PCR). Out of a total of 179 beef meat and meat contact surface samples, 83 (46.37%) were found to be contaminated with L. monocytogenes, with the highest incidence (53.01%) occurring in beef meat. In the present study, most of the isolated strains belonged to serogroups IIB and IVB (lineage I). The L. monocytogenes strain also contained monoA-B, prfA, plcA, plcB, mpl, hlyA, actA, gtcA, dltA, Fri, flaA, InlA, InlC, InlJ, and iap genes. Biofilm formation was not determined in the tested samples at pH 5.5 and different temperatures (4°C, 10°C, 25°C, and 37°C). However, strong biofilm formation was observed in 6.45% (2/31) of the strains at pH 7.0 after 48 h incubation at 37°C, and in 3.22% (1/31) of the strains at pH 7.0 after 48 h incubation at 4°C and 10°C. Pulsed-field gel electrophoresis (PFGE) results showed that L. monocytogenes isolates were clonally related, and cross-contamination was present. In addition, PFGE results also revealed that AscI had more distinguishing power than the ApaI restriction enzyme. These results indicate that L. monocytogenes detected from meat and meat contact surfaces in the slaughterhouse pose a potential risk to public health.

Molecular characterization of hybrid virulence plasmids in ST11-KL64 KPC-2-producing multidrug-resistant hypervirulent Klebsiella pneumoniae from China.

Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) strains combining virulence and multidrug resistance (MDR) features pose a great public health concern. The aim of this study is to explore the evolutionary characteristics of virulence in CR-HvKP by investigating the genetic features of resistance and virulence hybrid plasmids. The resistance and virulence phenotypes were determined by using antimicrobial susceptibility testing and the mouse bacteremia infection model, respectively. Plasmid profiles were investigated by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern blotting, conjugation assay, and whole genome sequencing (WGS). Bioinformatics tools were used to uncover the genetic features of the resistance and virulence hybrid plasmids. Two ST11-KL64 CRKP clinical isolates (KP18-3-8 and KP18-2079), which exhibited enhanced virulence compared with the classic CRKP, were detected positive for blaKPC-2 and rmpA2. The virulence level of the hypermucoviscous strain KP18-3-8 was higher than that of KP18-2079. S1-PFGE, Southern hybridization and WGS analysis identified two novel hybrid virulence plasmids in KP18-3-8 (pKP1838-KPC-vir, 228,158 bp) and KP18-2079 (pKP1838-KPC-vir, 182,326 bp), respectively. The IncHI1B/repB-type plasmid pKP1838-KPC-vir co-harboring blaKPC-2 and virulence genes (rmpA2, iucABCD and iutA) but lacking type IV secretion system could transfer into non-hypervirulent ST11 K. pneumoniae with the assistance of a helper plasmid in conjugation. The IncFII/IncR-type virulence plasmid pKP18-2079-vir may have been generated as a result of recombination between a typical pLVPK-like virulence plasmid and an MDR plasmid. Our studies further highlight co-evolution of the virulence and resistance plasmids in ST11-CRKP isolates. Close surveillance of such hybrid virulence plasmids in clinical K. pneumoniae should be performed.