Pulsed-field Gel Electrophoresis Typing 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.

Clone dissemination of IncX3 plasmid carrying blaNDM-1 in ST76 carbapenem resistance Klebsiella pneumoniae and bactericidal efficiency of aztreonam combined with avibactam in vitro and in vivo

ObjectivesThe rapid spread of the New Delhi Metal-β-lactamase-1 (NDM-1) gene in Klebsiella pneumoniae poses a substantial challenge to pediatric therapeutic care. Here, we aimed to characterise the IncX3-type plasmid carrying the blaNDM-1 gene in ST76 carbapenem resistance K. pneumoniae (CRKP) strains and assess the in vitro and in vivo bactericidal efficacy of Aztreonam (ATM) combined with Avibactam (AVI) (ATM+AVI) against CRKP. MethodsThe broth microdilution method and PCR were used to detect antimicrobial susceptibility and antibiotic resistance genes. Genetic relatedness was determined using Pulsed-Field Gel Electrophoresis (PFGE) and Multilocus Sequence Typing (MLST). The plasmid conjugation assay was used to verify the transmissibility of drug-resistant plasmids. Whole-Genome Sequencing (WGS) was employed to elucidate the genomic attributes of the genes. The Fractional Inhibitory Concentration (FIC) was calculated based on the checkerboard titration assay to determine the antimicrobial effect of ATM+AVI. The time-kill curve assay and a mouse anti-infection model were used to investigate the in vitro and in vivo bactericidal efficiency of ATM+AVI. ResultsSeven blaNDM-1-producing strains were found to be highly resistant to carbapenems, and they all belonged to the same sequence type (ST76) and were classified into the same PFGE clusters with an 89.1% similarity. The conjugation assay showed that the blaNDM-1-carrying plasmid was successfully transferred to Escherichia coli 600, resulting in transconjugants with carbapenem antibiotic resistance. A 54-kb IncX3 plasmid (pNDM-XZA88) carried the blaNDM-1 gene located on a Tn125 transposon-like element structure, demonstrating the transferability of resistance genes. Genome comparative analysis revealed that pNDM-XZA88 was highly similar to pCQ17 × 3 and pRor-30818cz and had relatively conserved backbones and variable accessory regions compared to the other four plasmids (pC39-334 kb, pNDM-1-DY1928, pNDM-K725, and pNDM-Z244). The checkerboard titration and time-kill curve assays revealed that the ATM+AVI combination therapy exerted significant bactericidal efficacy against the blaNDM-1-producing strains in vitro. The ATM+AVI combination also significantly reduced the bacterial burden in a mouse infection model constructed using the blaNDM-1-producing K. pneumoniae. ConclusionThis study demonstrated the clone dissemination of blaNDM-1-harboring IncX3 plasmids among the ST76 K. pneumoniae isolated from pediatric patients. Therefore, more attention should be paid to preventing this high-risk clone from harming pediatric patients. Moreover, we deduced that the ATM+AVI combination therapy is an effective strategy for treating blaNDM-1-producing K. pneumoniae.

Genetic approach toward linkage of Iran 2012–2016 cholera outbreaks with 7th pandemic Vibrio cholerae

Vibrio cholerae, as a natural inhabitant of the marine environment is among the world-leading causes of diarrheal diseases. The present study aimed to investigate the genetic relatedness of Iran 2012–2016 V. cholerae outbreaks with 7th pandemic cholera and to further characterize the non-ST69/non-ST75 sequence types strains by whole-genome sequencing (WGS).Twenty V. cholerae isolates related to 2012, 2013, 2015 and 2016 cholera outbreaks were studied by two genotyping methods – Pulsed-field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST)–and by antimicrobial susceptibility testing. Seven sequence types (STs) and sixteen pulsotypes were detected. Sequence type 69 was the most abundant ST confirming that most (65%, 13/20) of the studied isolates collected in Iran between 2012 and 2016 belonged to the 7th pandemic clone. All these ST69 isolates (except two) exhibited similar pulsotypes. ST75 was the second most abundant ST. It was identified in 2015 and 2016. ST438, ST178, ST579 and STs of 983 and 984 (as newfound STs) each were only detected in one isolate. All strains collected in 2016 appeared as distinct STs and pulsotypes indicative of probable different originations. All ST69 strains were resistant to nalidixic acid. Moreover, resistance to nalidixic acid, trimethoprim-sulfamethoxazole and tetracycline was only observed in strains of ST69. These properties propose the ST69 as a unique genotype derived from a separate lineage with distinct resistance properties. The circulation of V. cholerae ST69 and its traits in recent years in Iran proposes the 7th pandemic strains as the ongoing causes of cholera outbreaks in this country, although the role of ST75 as the probable upcoming dominant ST should not be ignored.Genomic analysis of non-ST69/non-ST75 strains in this study showed ST579 is the most similar ST type to 7th pandemic sequence types, due to the presence of wild type-El Tor sequences of tcpA and VC-1319, VC-1320, VC-1577, VC-1578 genes (responsible for polymyxin resistance in El Tor biotype), the traits of rstC of RS1 phage in one strain of this ST type and the presence of VPI-1 and VSP-I islands in ST579 and ST178 strains. In silico analysis showed no significant presence of resistance genes/cassettes/plasmids within non-ST69/non-ST75 strains genomes. Overall, these data indicate the higher susceptibility of V. cholerae non-ST69/non-ST75 strains in comparison with more ubiquitous and more circulating ST69 and ST75 strains.In conclusion, the occurrence of small outbreaks and sporadic cholera cases due to V. cholerae ST69 in recent years in Iran shows the 7th pandemic strains as the persistent causes of cholera outbreaks in this country, although the role of ST75 as the second most contributed ST should not be ignored. The occurrence of non-ST69/non-ST75 sequence types with some virulence factors characteristics in border provinces in recent years is noteworthy, and further studies together with surveillance efforts are expected to determine their likely route of transport.

Molecular characteristic of mcr-1 gene in Escherichia coli from aquatic products in Guangdong, China

Aquatic ecosystems serve as a dissemination pathway and a reservoir of both antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). This study aimed to determine the prevalence of colistin-resistant mcr-like genes in Enterobacteriales in aquatic products, which may be contribute to the transfer of ARGs in water environments. The mcr-1-positive Escherichia coli were recovered from 123 freshwater fish and 34 cultured crocodile cecum samples from 10 farmers' markets in Guangdong, China. Minimum inhibitory concentration (MIC) was determined using the agar dilution method. Genotyping was performed using pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Conjugation assay was carried out to investigate the transferability of mcr-1. Genomic information was obtained by whole genome sequencing (WGS) and bioinformatic analysis. Forty-four mcr-1 positive isolates showed co-resistance to tetracycline, trimethoprim/sulfamethoxazole, and gentamicin, while they were all sensitive to tigecycline, meropenem, and amikacin. They were typed into sixteen PFGE clusters. ST10 and ST117 were the most popular sequence types, followed by ST1114. S1-PFGE verified the presence of the mcr-1 gene on plasmids in sizes of ∼60 kb (n = 1) and ∼240 kb (n = 3). Whole genome sequencing-based analysis identified mcr-1 integrated in IncHI2 plasmid (n = 3), IncI2 plasmid (n = 2), and bacterial chromosome in two copies (n = 1). In addition to mcr-1, they carried several other antibiotic resistance genes, such as blaCTX-M-14, fosA3, and aac(6')-Ib-cr. These data suggest that aquatic products are an important antibiotic resistance reservoir and highlight possible risks regarding food safety.

Antimicrobial resistance in E. Coli of animal origin and discovery of a novel ICE mobile element in Northeast China

BackgroundMultidrug resistance in Enterobacteriaceae including resistance to quinolones is rising worldwide. The development of resistance may lead to the emergence of new transmission mechanisms. In this study, the collection of different E. coli was performed from animals and subjected to subsequent procedures including pulsed-field gel electrophoresis, micro-broth dilution method, polymerase chain reaction. Whole genome sequencing of E. coli C3 was performed to detect the affinity, antimicrobial resistance and major carriers of the isolates.ResultsA total of 66 E. coli were isolated and their antibiotic resistance genes, frequency of horizontal transfer and genetic environment of E. coli C3 were determined. The results showed there were both different and same types in PFGE typing, indicating clonal transmission of E. coli among different animals. The detection of antimicrobial resistance and major antibiotic resistance genes and the plasmid transfer results showed that strains from different sources had high levels of resistance to commonly used clinical antibiotics and could be spread horizontally. Whole-genome sequencing discovered a novel ICE mobile element.ConclusionIn summary, the antimicrobial resistance of E. coli in northeast China is a serious issue and there is a risk of antimicrobial resistance transmission. Meanwhile, a novel ICE mobile element appeared in the process of antimicrobial resistance formation.

Emergence of optrA-mediated linezolid resistance in clinical isolates of Enterococcus faecalis from Argentina

The aim of this study was to characterize the first 14 optrA-carrying linezolid resistant E. faecalis clinical isolates recovered in seven Argentinian hospitals between 2016 and 2021. The epidemiology of optrA-carrying isolates and the optrA genetic context were determined. The isolates were phenotypically and genotypically characterized. Susceptibility to 13 antimicrobial agents was performed; clonal relationship was assessed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Data provided by the whole-genome sequencing were used for identification of sequence types,antimicrobial resistancegenes, optrA variants, phylogenetic tree, and mobile genetic elements responsible to the dissemination of these strains. All the optrA-carrying E. faecalis isolates were multidrug-resistant and harboured several antimicrobial resistance genes. They carried three optrA variants and belonged to different lineages; however, three of them belonged to the hyperepidemic CC16. Mobile genetic elements were detected in all the isolates. The analysis of the optrA flanking region suggests the plasmidic localization in most of the isolates. To the best of our knowledge, this is the first report of optrA-mediated linezolid resistance in Argentina. The emergence and dissemination of the optrA genes in clinical E. faecalis isolates are of concern and highlights the importance of initiating the antimicrobial surveillance of Enterococcus spp. under a One Health strategy.

Comparative study on three molecular typing methods of Vibrio parahaemolyticus

To evaluate the correlation among the three molecular typing method of pulsed field gel electrophoresis(PFGE), repetitive extragenic palindromic(REP)-PCR and en-terobacterial repetitive intergenic consensus(ERIC)-PCR, and to explore the genetic relationship among strains, and to further understand the distribution and epidemic trend of Vibrio parahaemolyticus in Liaoning Province by combining Serotype analysis. Serum typing, PFGE, REP-PCR, and ERIC-PCR molecular typing and cluster analysis were performed on 150 VP isolates from Liaoning Province in 2018. 118 isolates could be divided into 14 Serotype, and 32 isolates could not be classified. The main serotypes were O3, O1 and O2. The resolution(DI) of PFGE is 0.969, the resolution(DI) of REP-PCR is 0.948, and the resolution(DI) of ERIC-PCR is 0.927. The Serotype O3 group strains are highly similar to the molecular types of O1 group strains. In 2018, the epidemic Serotype of clinical VP isolates in Liaoning Province is still O3: K6, and the epidemic serotype of food VP isolates is still O2. The result of PFGE, REP-PCR, and ERIC-PCR typing method are consistent, and the resolution and reproducibility of PFGE typing method are superior to the other two method. The Serotype O3 group is closely related to O1 group.

Within-Host Resistance and Virulence Evolution of a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae ST11 Under Antibiotic Pressure.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has recently aroused an extremely severe health challenge and public concern. However, the underlying mechanisms of fitness costs that accompany antibiotic resistance acquisition remain largely unexplored. Here, we report a hv-CRKP-associated fatal infection and reveal a reduction in virulence due to the acquisition of aminoglycoside resistance. The bacterial identification, antimicrobial susceptibility, hypermucoviscosity, virulence factors, MLST and serotypes were profiled.The clonal homology and plasmid acquisition among hv-CRKP strains were detected by XbaI and S1-PFGE. The virulence potential of the strains was evaluated using Galleria mellonella larvae infection model, serum resistance assay, capsular polysaccharide quantification, and biofilm formation assay. Genomic variations were identified using whole-genome sequencing (WGS). Four K. pneumoniae carbapenemase (KPC)-producing CRKP strains were consecutively isolated from an 86-year-old patient with severe pneumonia. Whole-genome sequencing (WGS) showed that all four hv-CRKP strains belonged to the ST11-KL64 clone. PFGE analysis revealed that the four ST11-KL64 hv-CRKP strains could be grouped into the same PFGE type. Under the pressure of antibiotics, the antimicrobial resistance of the strains increased and the virulence potential decreased. Further sequencing, using the Nanopore platform, was performed on three representative isolates (WYKP586, WYKP589, and WYKP594). Genomic analysis showed that the plasmids of these three strains underwent a large number of breaks and recombination events under antibiotic pressure. We found that as aminoglycoside resistance emerged via acquisition of the rmtB gene, the hypermucoviscosity and virulence of the strains decreased because of internal mutations in the rmpA and rmpA2 genes. This study shows that ST11-KL64 hv-CRKP can further evolve to acquire aminoglycoside resistance accompanied by decreased virulence to adapt to antibiotic pressure in the host.

Prevalence and Molecular Characterization of Shiga Toxin-Producing Escherichia coli from Food and Clinical Samples.

Shiga toxin-producing Escherichia coli (STEC) is one of the most prominent food-borne pathogens in humans. The current study aims to detect and to analyze the virulence factors, antibiotic resistance, and plasmid profiles for forty-six STEC strains, isolated from clinical and food strains. Pulsed-field gel electrophoresis (PFGE) was used to determine the genetic relatedness between different serotypes and sources of samples. The clinical samples were found to be resistant to Nb (100%), Tet (100%), Amp (20%), SXT (15%), and Kan (15%) antibiotics. In contrast, the food strains were found to be resistant to Nb (100%), Tet (33%), Amp (16.6%), and SXT (16.6%) antibiotics. The PFGE typing of the forty-six isolates was grouped into more than ten clusters, each with a similarity between 30% and 70%. Most of the isolates were found positive for more than five virulence genes (eae, hlyA, stx1, stx2, stx2f, stx2c, stx2e, stx2, nelB, pagC, sen, toxB, irp, efa, and efa1). All the isolates carried different sizes of the plasmids. The isolates were analyzed for plasmid replicon type by PCR, and 72.5% of the clinical isolates were found to contain X replicon-type plasmid, 50% of the clinical isolates contained FIB replicon-type plasmid, and 17.5% of the clinical isolates contained Y replicon-type plasmid. Three clinical isolates contained both I1 and Hi1 replicon-type plasmid. Only two food isolates contained B/O and W replicon-type plasmid. These results indicate that STEC strains have diverse clonal populations among food and clinical strains that are resistant to several antimicrobials. In conclusion, our findings indicate that food isolates of STEC strains harbor virulence, antimicrobial resistance, plasmid replicon typing determinants like those of other STEC strains from clinical strains. These results suggest that these strains are unique and may contribute to the virulence of the isolates. Therefore, surveillance and characterization of STEC strains can provide useful information about the prevalence of STEC in food and clinical sources. Furthermore, it will help to identify STEC serotypes that are highly pathogenic to humans and may emerge as a threat to public health.

Antibiotic resistance and genetic diversity among Pseudomonas aeruginosa isolated from urinary tract infections in Iran.

Aims: To determine the antibiotic resistance and genetic diversity of Pseudomonas aeruginosa isolates. Methods: The antibiotic resistance, genetic diversity and the conjugate transformation among Pseudomonas aeruginosa collected from patients with urinary tract infection in Tehran, Iran, wasinvestigated. Results: Antibiotic resistance against cefepime was seen in 51.74% of the isolates, followed by amikacin (47.76%). blaOXA-10 and blaVIM were the most prevalent extended-spectrum β-lactamaseand metallo-β-lactamases genes, respectively. Five clusters (C1-C5) were obtained by pulse field gel electrophoresis and multilocus sequence typing revealed two strain types, ST235 and ST664. Conjugation detected blaOXA-48 and blaNDM genes were transferred to Escherichia coli K12. Conclusion: The resistance of P. aeruginosa to antibiotics is increasing, which highlights the need to determine the resistance patterns to design better treatment strategies.

Molecular and Phenotypic Characterization of Ocular Methicillin-Resistant Staphylococcus epidermidis Isolates in Taiwan.

Staphylococcus epidermidis, a commensal, has emerged as an important opportunistic pathogen, particularly methicillin-resistant S. epidermidis (MRSE). The mechanism behind this transformation remains unclear. This study aimed to investigate the molecular and phenotypic characteristics of MRSE isolated from healthy conjunctiva and ocular infections. We collected MRSE isolates from two groups: healthy conjunctiva from patients undergoing cataract surgeries and ocular infections at our hospital. Genotypic analysis included pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), and biofilm-related genes (icaA, aap, and bhp). Additionally, phenotypic data on biofilm production and antibiotic susceptibility were recorded. A total of 86 isolates, including 42 from healthy conjunctiva and 44 from ocular infections, were analyzed. MLST identified 21 sequence types (STs), with ST59 being the most frequent (n = 33, 39.5%), followed by ST130 (n = 10, 11.6%), ST57 (n = 6, 7.0%), and ST2 (n = 6, 7.0%). All isolates were categorized in 23 PFGE types, and SCCmec IV was the most prevalent SCCmec type (n = 52, 60.5%). The two sources of isolates exhibited overlapping molecular types and phenotypic traits, although the ocular infection isolates exhibited significantly higher multidrug resistance compared to healthy conjunctiva isolates (P = 0.032). When contrasting ST59 with non-ST59, ST59 displayed a significantly higher presence of aap (100%) and bhp (69.7%) while lacking icaA (0%). ST59 also showed lower susceptibility to fluoroquinolones compared to non-ST59 (42.4%-54.5% vs. 75.5%-83.0%; P < 0.01). MRSE isolates from healthy conjunctiva and ocular infections demonstrated a degree of resemblance. Specific strains, notably ST59, exhibited distinctive characterizations.

Molecular Typing and Resistance Profile of Acinetobacter baumannii Isolates during the COVID-19 Pandemic: Findings from the "EPIRADIOCLINF" Project.

Due to the COVID-19 pandemic, there has been a shift in focus towards controlling the spread of SARS-CoV-2, which has resulted in the neglect of traditional programs aimed at preventing healthcare-associated infections and combating antimicrobial resistance. The present work aims to characterize the colonization or infection with Acinetobacter baumannii of COVID-19 patients and to identify any clonality between different isolates. Specifically, data and resistance profiles of A. baumannii isolates were prospectively collected from patients recruited by the EPIRADIOCLINF project. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were used for molecular typing. Overall, we analyzed 64 isolates of A. baumannii from 48 COVID-19 patients. According to our analysis, we have identified the spread of a clonally related isolate, referred to as B. The PFGE pattern B includes four subtypes: B1 (consisting of 37 strains), B2 (11), B3 (5), and B4 (2). Furthermore, in the isolates that were examined using MLST, the most observed sequence type was ST/281. In terms of resistance profiles, 59 out of the total isolates (92.2%) were found to be resistant to gentamicin, carbapenems, ciprofloxacin, and tobramycin. The isolation and identification of A. baumannii from COVID-19 patients, along with the high levels of transmission observed within the hospital setting, highlight the urgent need for the implementation of effective prevention and containment strategies.

Pulsed-field gel electrophoresis typing and molecular characterization of &lt;em&gt;Listeria monocytogenes&lt;/em&gt; isolates in raw milk samples from Polonnaruwa District, Sri Lanka

Listeria monocytogenes is an important food borne human pathogen associated with severe diseases in humans and animals. Human infection is associated with high mortality rates. Our previous studies done in 2012 on milk and milk products revealed 4b as the dominant serotype followed by 1/2a, 1/2b and 1/2c. Listeria monocytogenes is an important food borne human pathogen associated with severe diseases in humans and animals. Human infection is associated with high mortality rates. Our previous studies done in 2012 on milk and milk products revealed 4b as the dominant serotype followed by 1/2a, 1/2b and 1/2c. Therefore, this study was conducted to determine the sources of contamination and to identify the strain differences (pulsotypes) by pulsed-field gel electrophoresis (PFGE) typing and serotype profiles of L. monocytogenes isolates from farm collected raw milk samples from randomly identified locations in Grama Niladhari divisions of the Polonnaruwa district, Sri Lanka. Eighty isolates obtained from raw milk samples were confirmed as L. monocytogenes by Polymerase Chain Reaction, belonging to serotypes 4b, 1/2a, 1/2b and 1/2c. The strains were identified by PFGE. PFGE analysis digested with AscI and ApaI enzymes revealed different banding patterns. All the pulsotypes were found to be serotype 4b. The sub typing indicated the diversity of the Listeria species. The presence of serotypes 1/2a, 1⁄2b, 1/2c, and 4b in raw milk is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In our study, PFGE analysis allowed discrimination among isolates of the same serogroup. Further PFGE analysis showed heterogeneity among isolates recovered from both same sampling areas and different areas.

Extensive Expression of the Virulome Related to Antibiotic Genotyping in Nosocomial Strains of Klebsiella pneumoniae.

The emergence of hyper-virulent and multidrug-resistant (MDR) strains of Klebsiella pneumoniae isolated from patients with hospital- and community-acquired infections is a serious health problem that increases mortality. The molecular analysis of virulome expression related to antimicrobial-resistant genotype and infection type in K. pneumoniae strains isolated from patients with hospital- and community-acquired infections has been poorly studied. In this study, we analyzed the overall expression of the virulence genotype associated with the antimicrobial resistance genotype and pulse field gel electrophoresis (PFGE) type (PFtype) in K. pneumoniae. We studied 25 strains of K. pneumoniae isolated from patients who developed bacteremia and pneumonia during their hospital stay and 125 strains from outpatients who acquired community-acquired infections. Susceptibility to 12 antimicrobials was determined by Kirby-Bauer. The identification of K. pneumoniae and antibiotic-resistance genes was performed using polymerase chain reaction (PCR). To promote the expression of the virulence genes of K. pneumoniae, an in vitro infection model was used in human epithelial cell lines A549 and A431. Bacterial RNA was extracted with the QIAcube robotic workstation, and reverse transcription to cDNA was performed with the Reverse Transcription QuantiTect kit (Qiagen). The determination of the expression of the virulence genes was performed by real-time PCR. In addition, 57.3% (n = 86) of the strains isolated from patients with hospital- and community-acquired infections were multidrug-resistant (MDR), mainly to beta-lactam antibiotics (CB, AM, CFX, and CF), aminoglycosides (GE), quinolones (CPF and NOF), nitrofurantoin (NF), and sulfamethoxazole/trimethoprim (SXT). The most frequently expressed genes among strains isolated from hospital- and community-acquired infections were adhesion-type, ycfm (80%), mrkD (51.3%), and fimH (30.7%); iron uptake, irp2 (84%), fyuA (68.7%), entB (64.7%), and irp1 (56.7%); and protectins, rpmA (26%), which were related to antibiotic-resistance genes, blaTEM (96%), blaSHV (64%), blaCITM (52.6%), blaCTXM-1 (44.7%), tetA (74%), sul1 (57.3%), aac(3)-IV (40.7%), and aadA1 (36%). The results showed the existence of different patterns of expression of virulome related to the genotype of resistance to antimicrobials and to the PFtypes in the strains of K. pneumoniae that cause hospital- and community-acquired infections. These findings are important and may contribute to improving medical treatment strategies against infections caused by K. pneumoniae.

Secondary bacterial infections & extensively drug-resistant bacteria among COVID-19 hospitalized patients at the University Hospital in Kraków

IntroductionHealthcare-associated infections (HAI) and bacterial antimicrobial resistance posed a therapeutic risk during the coronavirus disease 2019 (COVID-19) pandemic. The aim of this study was to analyze the HAIs in COVID-19 patients in the Intensive Care Unit (ICU) and non-ICU at the University Hospital in Krakow (UHK) with an emphasis on the susceptibility of the most frequently isolated pathogens and the prevalence of extensively drug resistant (XDR) microorganisms.MethodsThis laboratory-based study was carried out at the University Hospital in Krakow in the ICU and non-ICUs dedicated to COVID-19 patients between May 2021 and January 2022. All isolates of Klebsiella pneumoniae were analyzed using PFGE protocol.Results292 independent HAI cases were identified, with the predominance of urinary tract infections (UTI), especially in the non-ICU setting. The most common ICU syndrome was pneumonia (PNA). The prevalence of XDR organisms was 22.6% in the ICU and 14.8% in non-ICUs among all isolates. The incidence of carbapenem-resistant Enterobacteriaceae infection was 24.8 cases per 10,000 hospitalizations and the carbapenem-resistant A. baumannii infection incidence was 208.8 cases per 10,000 hospitalizations. The prevalence of XDR strains was highest in Acinetobacter spp, in PNA cases. The PFGE typing demonstrated that almost all XDR strains varied widely from each other.ConclusionsIn this study, there was a high incidence of HAI in COVID-19 patients, especially when compared to Western Europe and the United States. Similarly, the prevalence of XDR microorganisms, especially XDR-A.baumannii, was also high. PFGE did not confirm the horizontal spread of any organism strains.

Validating the Utility of Multilocus Variable Number Tandem-repeat Analysis (MLVA) as a Subtyping Strategy to Monitor Listeria monocytogenes In-built Food Processing Environments

Listeria monocytogenes is a serious human pathogen and an enduring challenge to control for the ready-to-eat food processing industry. Cost-effective tools that can be deployed by commercial or in-house laboratories to rapidly investigate and resolve contamination events in the built food processing environment are of value to the food industry. Multilocus variable number tandem-repeat analysis (MLVA) is a molecular subtyping method, which along with other same-generation methods such as pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) is being superseded in disease tracking and outbreak investigations by whole-genome sequencing (WGS). In this paper, it is demonstrated that MLVA can continue to play a valuable role as a valid, fast, simple, and cost-effective method to identify and track Listeria monocytogenes subtypes in factory environments, with the method being highly congruent with MLST. Although MLVA does not have the discriminatory power of WGS to identify truly persistent clones, with careful interpretation of results alongside isolate metadata, it remains a powerful tool in situations and locations where WGS may not be readily available to food business operators.

A Burkholderia stabilis outbreak associated with the use of ultrasound gel in multiple healthcare centres in Montréal, Canada, May-October 2021.

Burkholderia stabilis is a non-fermenting, gram-negative bacteria that has previously been implicated in multiple nosocomial outbreaks through the use of contaminated medical devices and substances. This article reports on an outbreak of B. stabilis infections and colonizations, involving 11 patients from five acute care hospitals in Montréal, Canada. One sample was not available for testing, but the remaining 10 isolates (91%) were sent for phylogenetic testing. Medical materials and the patients' environments were also sampled and cultured. Samples were tested using pulsed field gel electrophoresis and multilocus sequence typing. The outbreak was found to be associated with the use of intrinsically contaminated non-sterile ultrasound gel. Relatedness of the gel's and the patients' B. stabilis strains was demonstrated using gel electrophoresis and multilocus sequence typing analyses. The investigation was concluded with a prompt recall of the product, and the outbreak was declared over by the end of October 2021. Contaminated non-sterile gel caused infections and pseudo-infections in several patients.

Molecular epidemiology, risk factors, and outcomes of carbapenem-resistant Klebsiella pneumoniae infection in a tertiary hospital in eastern China: for a retrospective study conducted over 4 years.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) have been extensively disseminated worldwide, resulting in increased mortality. We performed a retrospective analysis of the epidemiology and risk factors for the outcome of CRKP infection in a general teaching hospital in China. A molecular and clinical study was conducted for 98 CRKP in a tertiary hospital from January 2013 to December 2016. Carbapenemase gene detection, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were performed. Logistic regression was also used to identify the risk factors associated with 30-day mortality. The production of KPC carbapenemase was the main resistant mechanism, and KPC carbapenemase increased annually with a significant difference. However, the molecular outcome revealed the dominance and diversity in CRKP with 24 sequence types (STs) and 59 PFGE types (PTs). The ST11 CRKP strains, which showed a significant increasing trend year by year, were documented as predominant in our study. Additionally, the predominant ST11 CRKP corresponding to PT10 and PT15 continued to exhibit their characteristic patterns. Importantly, the newly identified PT09 and PT16 strains, corresponding to the ST11 lineage, were only discovered in 2016. Meanwhile, factors affecting 30-day mortality and ST11 proportionality with CRKP infection were assessed, and ST11, appropriate empirical treatment, and hospital stays were found to be independently associated with 30-day mortality. The ST11 CRKP strains played a dominant role in the process; however, the homology of these strains was polymorphic, and the advantage clusters were subject to changes through evolution. Furthermore, in addition to appropriate empirical treatment and hospital stays, ST11 CRKP was independently associated with 30-day mortality. To the best of our knowledge, this association was reported for the first time.

Using adenosine triphosphate bioluminescence level monitoring to identify bacterial reservoirs during two consecutive Enterococcus faecium and Staphylococcus capitis nosocomial infection outbreaks at a neonatal intensive care unit

IntroductionThis study aimed to assess the role of adenosine triphosphate (ATP) bioluminescence level monitoring for identifying reservoirs of the outbreak pathogen during two consecutive outbreaks caused by Enterococcus faecium and Staphylococcus capitis at a neonatal intensive care unit (NICU). The secondary aim was to evaluate the long-term sustainability of the infection control measures employed one year after the final intervention measures.MethodsTwo outbreaks occurred during a 53-day period in two disconnected subunits, A and B, that share the same attending physicians. ATP bioluminescence level monitoring, environmental cultures, and hand cultures from healthcare workers (HCW) in the NICU were performed. Pulsed-field gel electrophoresis (PFGE) typing was carried out to investigate the phylogenetic relatedness of the isolated strains.ResultsFour cases of E. faecium sepsis (patients A-8, A-7, A-9, B-8) and three cases of S. capitis sepsis (patients A-16, A-2, B-8) were diagnosed in six preterm infants over a span of 53 days. ATP levels remained high on keyboard 1 of the main station (2076 relative light unit [RLU]/100 cm2) and the keyboard of bed A-9 (4886 RLU/100 cm2). By guidance with the ATP results, environmental cultures showed that E. faecium isolated from the patients and from the main station’s keyboard 1 were genotypically indistinguishable. Two different S. capitis strains caused sepsis in three patients. A total 77.8% (n = 7/9) of S. capitis cultured from HCW's hands were genotypically indistinguishable to the strains isolated from A-2 and A-16. The remaining 22.2% (n = 2/9) were genotypically indistinguishable to patient B-8. Three interventions to decrease the risk of bacterial transmission were applied, with the final intervention including a switch of all keyboards and mice in NICU-A and B to disinfectable ones. Post-intervention prospective monitoring up to one year showed a decrease in blood culture positivity (P = 0.0019) and catheter-related blood stream infection rate (P = 0.016) before and after intervention.ConclusionATP monitoring is an effective tool in identifying difficult to disinfect areas in NICUs. Non-medical devices may serve as reservoirs of pathogens causing nosocomial outbreaks, and HCWs' hands contribute to bacterial transmission in NICUs.

Characterization of Leuconostoc carnosum and Latilactobacillus sakei during Cooked Pork Ham Processing

Cooked ham is a popular, ready-to-eat product made of pork meat that is susceptible to microbial growth throughout its shelf life. In this study, we aimed to monitor the microbial growth and composition of nine vacuum-packed cooked ham lots using plate counting until the microbial limit of 7.4 log10 AMC/LAB CFU/g was exceeded. Eight out of nine lots exceeded the microbial limit after 20 days of storage. Lactic acid bacteria strains, particularly Leuconostoc carnosum and Latilactobacillus sakei, prevailed in vacuum-packed cooked ham. Leuconostoc carnosum 2 (Leuc 2) and Latilactobacillus sakei 4 (Sakei 4) were isolated from raw meat and the post-cooking area of the food processing facility. Carbohydrate utilization patterns of Leuc. carnosum PFGE types isolated from raw meat and the food processing environment differed from those isolated from cooked ham. These findings demonstrate how raw meat and its processing environment impact the quality and shelf life of cooked ham.