Pulsed-field Gel Electrophoresis Research Articles

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.

Determination of Capsule Types, Antibiotic Resistance Profiles and Phylogenetic Relationships of Group B Streptococcus Isolates Isolated from Patients Followed in Various Clinics and Polyclinics

Group B streptococci (GBS) are microorganisms that cause various systemic infections. In this study, it was aimed to investigate the capsule serotypes, antibiotic resistance and phylogenetic similarity relationship between GBS isolates. One hundred and ten GBS isolates isolated from female patients who admitted to Adana City Hospital with various complaints were included in the study. Kirby-Bauer disc diffusion method was used for the antibiotic resistance patterns and evaluated with CLSI criteria. The genes ermB, ermTR, mefA for erythromycin resistance and linB genes for clindamycin resistance were investigated by multiplex PCR method. Multiplex PCR method was used for GBS capsule serotyping. Similarity relationship between the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) method. As a result of the study; all strains were found to be sensitive to penicillin and vancomycin. Erythromycin, clindamycin ofloxacin, and ceftriaxone resistance rates were observed as 60%, 11.8%, 6.4%, and 4.5%, respectively. The mefA gene was not found while 53% and 47% of the erythromycin-resistant isolates carried ermTR and ermB genes, respectively. The linB gene was not found in clindamycin-resistant GBS isolates. The capsule serotype distributions of GBSs were found as, Ib 42.7%, Ia 35.5%, III 10%, II 8.2%, and V 3.6%, respectively. In the analysis of the similarity relationships between GBS isolates with the PFGE method, no significant relationship was found. In conclusion, it was thought that more studies should be conducted to show the prevalence of GBS capsule serotypes and patterns of antibiotic resistance.

Gut colonization and subsequent infection of neonates caused by extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae.

The gut microbiota harbors diverse bacteria considered reservoirs for antimicrobial resistance genes. The global emergence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacterales (ESBL-PE) significantly contributes to healthcare-associated infections (HAIs). We investigated the presence of ESBL-producing Escherichia coli (ESBL-PEco) and ESBL-producing Klebsiella pneumoniae (ESBL-PKpn) in neonatal patients' guts. Furthermore, we identified the factors contributing to the transition towards ESBL-PEco and ESBL-PKpn-associated healthcare-associated infections (HAIs). The study was conducted from August 2019 to February 2020, in a Neonatal Intensive Care Unit of the Hospital Infantil de México Federico Gómez. Rectal samples were obtained upon admission, on a weekly basis for a month, and then biweekly until discharge from the neonatology ward. Clinical data, culture results, and infection information were gathered. We conducted antimicrobial tests, multiplex PCR assay, and pulsed-field gel electrophoresis (PFGE) to determine the antimicrobial resistance profile and genetic relationships. A comparison between the group's controls and cases was performed using the Wilcoxon and Student t-tests. Of the 61 patients enrolled, 47 were included, and 203 rectal samples were collected, identifying 242 isolates. In 41/47 (87%) patients, colonization was due to ESBL-PEco or ESBL-PKpn. And nine of them developed HAIs (22%, 9/41). ESBL-PEco resistance to cephalosporins ranged from 25.4% to 100%, while ESBL-PKpn resistance varied from 3% to 99%, and both bacteria were susceptible to carbapenems, tigecillin, and colistin. The prevalent bla CTX-M-group-1 gene accounted for 77.2% in ESBL-PEco and 82.2% in ESBL-PKpn, followed by bla TEM 50% and bla OXA-1 43.8% in ESBL-PEco and bla TEM 80.2% and bla SHV 76.2% in ESBL-PKpn. Analysis of clonality revealed identical colonizing and infection isolates in only seven patients. Significant risk factors included hospital stay duration, duration of antibiotic treatment, and invasive device usage. Our findings suggest high ESBL-PEco and ESBL-PKpn rates of colonization often lead to infection in neonates. Attention should be paid to patients with ESBL-PE.

Pathogenic Potential of Coagulase-Positive Staphylococcus Strains Isolated from Aviary Capercaillies and Free-Living Birds in Southeastern Poland.

The aim of the study was to determine the occurrence and characteristics of coagulase-positive Staphylococcus strains in the carcasses of wild birds and aviary capercaillies in Southeastern Poland. In total, samples taken from 333 birds were examined. The material consisted of swabs from the internal organs of dead birds (heart, liver, and spleen), the tarsal joints, and mucous membranes (conjunctiva and palatine fissure), as well as from unhatched embryos. The isolated Staphylococcus strains were tested for sensitivity to nine antimicrobial agents and the presence of selected virulence genes. An analysis of the similarity of isolates within species was performed using pulsed-field gel electrophoresis (PFGE). The result indicates that coagulase-positive strains accounted for 5.7% and belonged to the species: Staphylococcus aureus, Staphylococcus pseudintermedius, and Staphylococcus delphini. Among isolated strains, 15.8% were multidrug resistant. The most frequently detected virulence genes were hla in 58% of isolates and hlb and hld in 47.4% of isolates. The results of multiplex PCR showed the presence of genes responsible for the production of enterotoxins C, B, E, and J, in single isolates. It can be concluded that coagulase-positive Staphylococcus strains accounted for a small percentage of staphylococci isolated from free-living birds in the study area. The occurrence of multidrug-resistant coagulase-positive Staphylococcus strains in aviary capercaillies suggests that they play a role in the transmission and spread of resistant strains into the environment. Free-living birds may also be a reservoir of enterotoxigenic Staphylococcus strains.

Phylogenetic Aspects of Antibiotic Resistance and Biofilm Formation of P. aeruginosa Isolated from Clinical Samples.

Biofilm production and drug resistance phenomenon play a critical role in P. aeruginosa infections. Several genes, including psl, pel, brlR, and mex, are involved in the phenomenon. The aim of this study was to find the relationship between the mentioned genes and the sources of P. aeruginosa infections. Fifty-nine P. aeruginosa isolates detected from clinical specimens were used to determine antibiotic susceptibility patterns, prevalence of the genes using PCR, biofilm formation, biofilm eradication concentration assay (MBEC), and epidemiological characteristics using pulsed-field gel electrophoresis (PFGE). The results showed that 35.6% and 16.94% of all the samples were isolated from urine and wounds, 81.33% of the isolates were biofilm producers, 27.11% were multidrug-resistant (MDR), and 100% of the main biofilm former genes belonged to pslA. 94.91% of the isolates possessed brlR and mexA, and 91.5% of them expressed pslA. It was also indicated that neither ciprofloxacin nor imipenem could eradicate the formed biofilms. Moreover, we could identify 81.4% distinctive restriction profiles among the isolates, using an 80% similarity cutoff point; brlR and pel genes were significantly (P=0.032; P=0.044) related to phylogenetic pulsotypes. Comparison of the dendrogram in the isolates revealed that the detected isolates from urine were present in 12 different pulsotypes. It was found that there was a relationship between MDR, biofilm production, and brlR and pel genes among the isolates. It is distinguished there were similar genetic patterns between detected isolates from urine and could be concluded that the urinary tract played a critical role in maintaining and transferring biofilm drug-resistant genes of P. aeruginosa in clinical sites. The study highlights the importance of urine in distribution of clinical biofilm formation and drug-resistant P. aeruginosa isolates.

Different cellular and molecular responses of Bovine milk phagocytes to persistent and transient strains of Streptococcus uberis causing mastitis.

Streptococcus uberis is frequently isolated from milk collected from dairy cows with mastitis. According to the host's immunity, bacterial virulence, and their interaction, infection with some strains can induce persistent subclinical inflammation, while infection with others induces severe inflammation and transient mastitis. This study compared the inflammatory response of milk-isolated white blood cells (mWBCs) to persistent and transient S. uberis strains. Quarter milk samples were collected aseptically for bacterial culture from all lactating cows once a week over a 10-week period. A transient and noncapsular strain with a 1-week intramammary infection duration was selected from this herd, while a persistent and capsular S. uberis strain with an intramammary infection longer than 2 months from our previous study was selected based on an identical pulse field gel electrophoresis pattern during the IMI episode. Cellular and molecular responses of mWBCs were tested, and the data were analyzed using repeated analysis of variance. The results showed a higher response in migration, reactive oxygen species generation, and bacterial killing when cells were stimulated with transient S. uberis. In contrast, the persistent strain led to increased neutrophil extracellular trap release. This study also highlighted several important molecular aspects of mWBCs. Gene expression analyses by real-time RT-PCR revealed a significant elevation in the expression of Toll-like receptors (TLR-1, TLR-2, TLR-6) and proinflammatory cytokines (tumor necrosis factor-alpha or TNF-α) with the transient strain. Additionally, Streptococcus uberis capsule formation might contribute to the capability of these strains to induce different immune responses. Altogether, these results focus on the immune function of activated mWBCs which demonstrate that a transient strain can elicit a stronger local immune response and, subsequently, lead to rapid recovery from mastitis.

Concurrent transmission of multiple carbapenemases in a long-term acute-care hospital.

We investigated concurrent outbreaks of Pseudomonas aeruginosa carrying blaVIM (VIM-CRPA) and Enterobacterales carrying blaKPC (KPC-CRE) at a long-term acute-care hospital (LTACH A). We defined an incident case as the first detection of blaKPC or blaVIM from a patient's clinical cultures or colonization screening test. We reviewed medical records and performed infection control assessments, colonization screening, environmental sampling, and molecular characterization of carbapenemase-producing organisms from clinical and environmental sources by pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing. From July 2017 to December 2018, 76 incident cases were identified from 69 case patients: 51 had blaKPC, 11 had blaVIM, and 7 had blaVIM and blaKPC. Also, blaKPC were identified from 7 Enterobacterales, and all blaVIM were P. aeruginosa. We observed gaps in hand hygiene, and we recovered KPC-CRE and VIM-CRPA from drains and toilets. We identified 4 KPC alleles and 2 VIM alleles; 2 KPC alleles were located on plasmids that were identified across multiple Enterobacterales and in both clinical and environmental isolates. Our response to a single patient colonized with VIM-CRPA and KPC-CRE identified concurrent CPO outbreaks at LTACH A. Epidemiologic and genomic investigations indicated that the observed diversity was due to a combination of multiple introductions of VIM-CRPA and KPC-CRE and to the transfer of carbapenemase genes across different bacteria species and strains. Improved infection control, including interventions that minimized potential spread from wastewater premise plumbing, stopped transmission.

Characterization of nontyphoidal Salmonella strains from a tertiary hospital in China: serotype diversity, multidrug resistance, and genetic insights.

Nontyphoidal Salmonella is a significant public health concern due to its ability to cause foodborne illnesses worldwide. This study aims to characterize the nontyphoidal Salmonella strains isolated from patients in China. A total of 19 nontyphoidal Salmonella strains were characterized through serovar identification, antimicrobial susceptibility testing (AST), biofilm formation assessment. Genetic relatedness was determined using pulsed-field gel electrophoresis (PFGE). WGS was employed to decipher the resistance mechanism and to contextualize the S. serovar Mbandaka strains among previously sequenced isolates in China. The biofilm associated mrkA gene was examined by PCR. The predominant serovar identified was S. Enteritidis, followed by S. Mbandaka, S. Thompson, S. Livingston, S. Alachua, and S. Infantis. PFGE analysis indicated a notable genetic similarity among the S. Mbandaka isolates. Phylogenetic analysis suggested that these strains were likely derived from a single source that had persisted in China for over five years. One multidrug resistance (MDR) S. Enteritidis isolate carried a highly transferable IncB/O/K/Z plasmid with bla CTX-M-15. One S. Thompson strain, harboring the mrkABCDF operon in an IncX1 plasmid, isolated from cutaneous lesions, demonstrated robust biofilm formation. However, no mrkABCDF loci were detected in other strains. Our study emphasizes the importance of persisted surveillance and prompt response to Salmonella infections to protect public health. The dissemination of bla CTX-M-15-harboring IncB/O/K/Z plasmid and the spread of virulent mrkABCDF operon among Salmonella in China and other global regions warrant close monitoring.

Foodborne Outbreak by Salmonella enterica Serovar Weltevreden in West Bengal, India.

Foodborne gastroenteritis outbreaks owing to Salmonella enterica serovar Weltevreden (Salmonella Weltevreden) represent a significant global public health problem. In the past two decades, Salmonella Weltevreden has emerged as a dominant foodborne pathogen, especially in South-East Asian countries. This report describes a community foodborne outbreak of gastroenteritis caused by Salmonella Weltevreden in August 2022 following consumption of panipuri from a street vendor in the Polba block in Hooghly district, West Bengal, India. This food item was consumed by 185 people, of whom 129 had acute watery diarrhea with other clinical symptoms and 65 of them were admitted to different District hospitals for treatment. Stool specimens collected from hospitalized cases were positive for S. enterica, and further serotyped as Salmonella Weltevreden. All the Salmonella Weltevreden strains possessed the Salmonella pathogenicity islands associated genes (invA/E, orgA, ttrc, ssaQ, mgtC, misL, spi4D), the enterotoxin (stn), and hyperinvasive locus gene (hilA). Except erythromycin, all the strains were susceptible for commonly used antimicrobials in the treatment of diarrhea. The XbaI-based pulsed-field gel electrophoresis analysis indicated that all the isolates responsible for the recent outbreak were similar, but diverged from other Salmonella Weltevreden that were previously reported in West Bengal. This report indicates that foodborne infection is a major public health concern in India and demands to strengthen capacity-building measures at the local health care levels for linking causative agents of outbreaks.

A Food Poisoning Caused by Salmonella Enterica (S. Enteritidis) ST11 Carrying Multi-Antimicrobial Resistance Genes in 2019, China.

This study was to identify and analyze the pathogen responsible for food poisoning in a tourist group traveling from Macao to Zhuhai. Samples were obtained from 27 patients of 96 cases, as well as samples of contaminated food in Macau. The collected samples were subjected to serological identification, drug sensitivity analysis, drug resistance gene identification, virulence factor analysis, and tracing. Twenty-six isolates and the salad isolate were S. enteritidis ST11. Isolates from patients were exhibited significant resistance to Penicillin AMP (Ampicillin) and quinolones NAL (Nalidixic acid). Among these isolates, 21 strains were resistant to two or more antibiotics, indicating the multi-drug resistance (MDR). Genomic characteristics and phylogenetic analysis were performed on 9 of the isolates using whole genome sequencing (WGS). The analysis revealed that the resistance to AMP and NAL was primarily caused by a gryA mutation D87Y (9/9, 100%), and the presence of beta-lactam resistance genes blaOXA-1 (1/9, 11.11%), blaTEM-141 (1/9, 11.11%), and blaTEM-1B (8/9, 88.89%). It was also found a strains isolated from patients had two resistance genes to quinolones or beta-lactam drugs (1/8, 12.5%), respectively. The strains were found to possess 165 virulence genes, one adherence class virulence factor, one invasion class virulence factor and various pathogenicity islands, including SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-9, SPI-10, SPI-13, SPI-14, SPI-15, SGI 1, CS54_island, and C63PI-1. Additionally, the virulence plasmids were detected, including IncFIB(s)-IncFII(s)-IncX1 (55.56%), IncFIB(s)-IncFII(s) (33.33%), and IncFIB(s)-IncFII(s)-IncHI2-IncHI2A (11.11%). PFGE (Pulsed Field Gel Electrophoresis) and phylogenetic tree analysis revealed a high degree of similarity between Salmonella isolates from patients and food samples from Macao. This study identified Salmonella enterica ST11 as the cause of the food poisoning outbreak. The findings highlight the importance of phenotypic characterization and next-generation sequencing (NGS) tools in epidemiological studies and emphasize the potential risk of a new emerging multi-antibiotic ST11 clone for S. enteritidis.

Analysis of in vitro expression of virulence genes related to antibiotic and disinfectant resistance in Escherichia coli as an emerging periodontal pathogen.

The collective involvement of virulence markers of Escherichia coli as an emerging pathogen associated with periodontitis remains unexplained. This study aimed to implement an in vitro model of infection using a human epithelial cell line to determine the virulome expression related to the antibiotic and disinfectant resistance genotype and pulse field gel electrophoresis (PFGE) type in E. coli strains isolated from patients with periodontal diseases. We studied 100 strains of E. coli isolated from patients with gingivitis (n = 12), moderate periodontitis (n = 59), and chronic periodontitis (n = 29). The identification of E. coli and antibiotic and disinfectant resistance genes was performed through PCR. To promote the expression of virulence genes in the strains, an in vitro infection model was used in the human epithelial cell line A549. RNA was extracted using the QIAcube robotic equipment and reverse transcription to cDNA was performed using the QuantiTect reverse transcription kit (Qiagen). The determination of virulence gene expression was performed through real-time PCR. Overall, the most frequently expressed adhesion genes among the isolated strains of gingivitis, moderate periodontitis, and chronic periodontitis were fimH (48%), iha (37%), and papA (18%); those for toxins were usp (33%); those for iron acquisition were feoB (84%), fyuA (62%), irp-2 (61%), and iroN (35%); those for protectins were traT (50%), KpsMT (35%), and ompT (28%); and those for pathogenicity islands were malX (45%). The most common antibiotic and disinfectant resistance genes among gingivitis, moderate periodontitis, and chronic periodontitis strains were sul-2 (43%), blaSHV (47%), blaTEM (45%), tet(A) (41%), dfrA1 (32%), marR-marO (57%), and qacEA1 (79%). The findings revealed the existence of a wide distribution of virulome expression profiles related to the antibiotic and disinfectant resistance genotype and PFGE type in periodontal strains of E. coli. These findings may contribute toward improving the prevention and treatment measures for periodontal diseases associated with E. coli.

Puerperal mastitis caused by limited community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) clones.

To outline the epidemiology of puerperal mastitis caused by methicillin-resistant Staphylococcus aureus (MRSA) and evaluate the effect of an infection control bundle on its incidence. A surge in MRSA puerperal mastitis was noted in a community hospital in September 2009. MRSA samples from mastitis cases and the environment underwent typing using multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), gene encoding surface protein A (spa), accessory gene regulator (agr), and pulsed-field gel electrophoresis (PFGE). The phenotypic characteristics, including superantigen toxin profiles, gene encoding Panton-Valentine leucocidin (pvl), and minimal inhibitory concentration (MIC) against vancomycin, were ascertained. Subsequently, an infection control bundle emphasizing contact precautions was introduced, and mastitis incidence rates pre- and post-intervention were compared. The majority of cases occurred within 6 weeks post-delivery in first-time mothers. Of the 42 S. aureus isolates (27 from mastitis and 15 from colonized staff and environmental sources), 25 (92.6%) clinical and 3 (20%) colonized MRSA were identified as ST59-SCCmecVT-spa t437-agr group I with a vancomycin MIC of 1 mg/L, pvl-positive, and predominantly with a consistent toxin profile (seb-selk-selr). PFGE revealed 13 patterns; pulsotype B exhibited clonal relatedness between two clinical and three colonized MRSA samples. Post-intervention, the incidence of both mastitis and MRSA mastitis notably decreased from 13.01 to 1.78 and from 3.70 to 0.99 episodes per 100 deliveries, respectively. Distinct community-associated MRSA (CA-MRSA) clones were detected among puerperal mastitis patients and colonized staff. The outbreak was effectively controlled following the implementation of a targeted infection control bundle.

ST11 KPC-2-Producing Klebsiella pneumoniae Isolated from Patient with Acute Myelocytic Leukemia.

The emergence of the ST11-CRKP (ST11-CRKP) strain is expected to become a serious public health problem in China. As one of the most serious complications in patients with acute myeloid lymphoma, infections can cause systemic infection and life-threatening sepsis, seriously affecting the morbidity, mortality, and quality of life of patients. Thus, ST11-CRKP infections in patients with acute myeloid lymphoma are worthy of our attention. To investigate the occurrence and genetic characteristics of the ST11-CRKP from a patient with acute myeloid lymphoma. Species identification was determined by MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was conducted by VITEK 2 system with AST-N335 panel. Whole-genome sequencing was performed on the Illumina NovaSeq 6000 platform. Phylogenetic analyses were performed using Snippy based on the core-genome SNPs. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot and Whole-genome analysis indicated blaKPC-2 genes were located on plasmids with a conserved genetic environment. Moreover, the eight ST11-CRKP strains carry a variety of antimicrobial resistance genes (ARGs) and virulence factors. The ability of biofilm formation of eight strains was verified by a crystal violet assay. Core genome single-nucleotide polymorphism (cgSNP) analysis suggesting a possible bacterial translocation event. We performed a comprehensive analysis of ST11-CRKP strains from a patient with acute myelocytic leukemia. Our study emphasized the need for continuous surveillance of ST11-CRKP in the clinic especially in the immunocompromised population.

Carbapenemase-producing Enterobacterales isolated from hospital sinks: molecular relationships with isolates from patients and the change in contamination status after daily disinfection with sodium hypochlorite.

This study aimed to investigate the contamination status of hospital sinks with carbapenemase-producing Enterobacterales (CPE), the efficacy of daily cleaning with sodium hypochlorite, and the relationships between CPEs isolated from contaminated sinks and patients. Pre/postintervention surveys of the CPE-contaminated sinks. Hospital wards including pediatric intensive care unit in a children's hospital. Consenting CPE-colonized patients admitted between November 2018 and June 2021 in our hospital. Environmental culture of 180 sinks from nine wards in our hospital was performed three times with an interval of 2 years (2019, 2021, 2023). Molecular typing of the isolated strains from the sinks and patients was performed. After the first surveillance culture, we initiated daily disinfection of the sinks using sodium hypochlorite. Before the intervention, we detected 30 CPE-positive sinks in 2019. After the intervention with sodium hypochlorite, we observed a substantial decline in the number of sinks contaminated with CPE; 13 in 2021 and 6 in 2023. However, the intervention did not significantly reduce the number of CPE-contaminated sinks used for the disposal of nutrition-rich substances. The CPE isolates from the patients and those from the sinks of the wards or floors where they were admitted tended to have similar pulse-field gel electrophoresis patterns. Contaminated sinks could be reservoirs of disseminating CPE to the patients. Daily disinfection of sinks with sodium hypochlorite may be effective in eliminating CPE, although the effect could be weaker in sinks with a greater risk of contact with nutrition-rich substances.

Biodiversity and Safety: Cohabitation Experimentation in Undefined Starter Cultures for Traditional Dairy Products

Natural starter cultures, characterised by undefined microbiota, can contribute to the technological process, giving peculiar characteristics to artisanal fermented foods. Several species have a long history of safe use and have obtained Qualified Presumption of Safety (QPS) status from the European Food Safety Authority (EFSA), whereas others (non-QPS) could represent a potential risk for consumers’ health and must undergo a safety assessment. In this work, the biodiversity, at species and strain level, by pulsed-field gel electrophoresis (PFGE) and (GTG)5 rep-PCR, of an undefined natural starter culture, in frozen and lyophilized form, obtained from ewe’s raw milk avoiding thermal treatment or microbial selection, was investigated. The culture was constituted by different biotypes of Enterococcus durans, Enterococcus faecium, Enterococcus faecalis, and Lacticaseibacillus paracasei. Streptococcus oralis and Streptococcus salivarius were also found, over species belonging to the Streptococcus bovis–Streptococcus equinus complex (SBSEC), like Streptococcus gallolyticus subsp. macedonicus, Streptococcus lutetiensis, and Streptococcus equinus. Molecular investigation on virulence and antibiotic resistance genes, as well as minimum inhibitory concentration (MIC) determination, revealed that all the non-QPS strains can be considered safe in the perspective of using this culture for cheesemaking. The obtainment of a natural culture directly from ewe’s raw milk bypassing thermal treatment and selection of pro-technological bacteria can be advantageous in terms of biodiversity preservation, but non-QPS microorganisms can be included in the natural starter and also in cheeses, especially in traditional ones obtained from fermenting raw milk. Following EFSA guidelines, artisanal factories should not be allowed to produce starter cultures by themselves from raw milk, running the risk of including some non-QPS species in their culture, and only selected starters could be used for cheesemaking. A revision of the criteria of QPS guidelines should be necessary.

Sink-traps are a major source for carbapenemase-producing Enterobacteriaceae transmission.

We studied the extent of carbapenemase-producing Enterobacteriaceae (CPE) sink contamination and transmission to patients in a nonoutbreak setting. During 2017-2019, 592 patient-room sinks were sampled in 34 departments. Patient weekly rectal swab CPE surveillance was universally performed. Repeated sink sampling was conducted in 9 departments. Isolates from patients and sinks were characterized using pulsed-field gel electrophoresis (PFGE), and pairs of high resemblance were sequenced by Oxford Nanopore and Illumina. Hybrid assembly was used to fully assemble plasmids, which are shared between paired isolates. In total, 144 (24%) of 592 CPE-contaminated sinks were detected in 25 of 34 departments. Repeated sampling (n = 7,123) revealed that 52%-100% were contaminated at least once during the sampling period. Persistent contamination for >1 year by a dominant strain was common. During the study period, 318 patients acquired CPE. The most common species were Klebsiella pneumoniae, Escherichia coli, and Enterobacter spp. In 127 (40%) patients, a contaminated sink was the suspected source of CPE acquisition. For 20 cases with an identical sink-patient strain, temporal relation suggested sink-to-patient transmission. Hybrid assembly of specific sink-patient isolates revealed that shared plasmids were structurally identical, and SNP differences between shared pairs, along with signatures for potential recombination events, suggests recent sharing of the plasmids. CPE-contaminated sinks are an important source of transmission to patients. Although traditionally person-to-person transmission has been considered the main route of CPE transmission, these data suggest a change in paradigm that may influence strategies of preventing CPE dissemination.

A cluster of three extrapulmonary Mycobacterium abscessus infections linked to well-maintained water-based heater-cooler devices.

Various water-based heater-cooler devices (HCDs) have been implicated in nontuberculous mycobacteria outbreaks. Ongoing rigorous surveillance for healthcare-associated M. abscessus (HA-Mab) put in place following a prior institutional outbreak of M. abscessus alerted investigators to a cluster of 3 extrapulmonary M. abscessus infections among patients who had undergone cardiothoracic surgery. Investigators convened a multidisciplinary team and launched a comprehensive investigation to identify potential sources of M. abscessus in the healthcare setting. Adherence to tap water avoidance protocols during patient care and HCD cleaning, disinfection, and maintenance practices were reviewed. Relevant environmental samples were obtained. Patient and environmental M. abscessus isolates were compared using multilocus-sequence typing and pulsed-field gel electrophoresis. Smoke testing was performed to evaluate the potential for aerosol generation and dispersion during HCD use. The entire HCD fleet was replaced to mitigate continued transmission. Clinical presentations of case patients and epidemiologic data supported intraoperative acquisition. M. abscessus was isolated from HCDs used on patients and molecular comparison with patient isolates demonstrated clonality. Smoke testing simulated aerosolization of M. abscessus from HCDs during device operation. Because the HCD fleet was replaced, no additional extrapulmonary HA-Mab infections due to the unique clone identified in this cluster have been detected. Despite adhering to HCD cleaning and disinfection strategies beyond manufacturer instructions for use, HCDs became colonized with and ultimately transmitted M. abscessus to 3 patients. Design modifications to better contain aerosols or filter exhaust during device operation are needed to prevent NTM transmission events from water-based HCDs.

Molecular characterization of the multi-drug resistant Myroides odoratimimus isolates: a whole genome sequence-based study to confirm carbapenem resistance.

The bacteria belonging to the Myroides genus are opportunistic pathogens causing community or hospital-acquired infections that result in treatment failure due to antibiotic resistance. This study aimed to investigate molecular mechanisms of antibiotic resistance, clonal relatedness, and the biofilm forming capacity of the 51 multi-drug resistant Myroides odoratimimus. All isolates were screened for blaKPC, blaOXA, blaVIM, blaIMP, blaMUS, blaTUS, blaNDM, and blaB genes by using PCR amplification. Whole genome sequencing (WGS) was applied on three randomly selected isolates for further investigation of antibiotic resistance mechanisms. Clonal relatedness was analyzed by Pulsed-field gel electrophoresis (PFGE) and the microtiter plate method was used to demonstrate biofilm formation. All isolates were positive for biofilm formation. PCR analysis resulted in a positive for only the blaMUS-1 gene. WGS identified blaMUS-1, erm(F), ere(D), tet(X), and sul2 genes in all strains tested. Moreover, the genomic analyses of three strains revealed that genomes contained a large number of virulence factors (VFs). PFGE yielded a clustering rate of 96%. High clonal relatedness, biofilm formation, and multi-drug resistance properties may lead to the predominance of these opportunistic pathogens in hospital environments and make them cause nosocomial infections.

Exploring the genetic diversity of Edwardsiella ictaluri in Vietnamese striped catfish (Pangasianodon hypophthalmus) farms over a 20-year period

Since first being identified in 1999, disease outbreaks from Edwardsiella ictaluri remain a significant health challenge for the farmed catfish sector in Vietnam. To better understand the population structure of E. ictaluri circulating in Vietnamese striped catfish (Pangasianodon hypophthalmus) farms, this study aimed to investigate the genetic diversity of 83 E. ictaluri isolates recovered from natural disease outbreaks occurring in the Mekong Delta region between 2001 and 2021. Pulsed-field gel electrophoresis resolved isolates into 15 pulsotypes following restriction digest with speI, with a Simpson’s diversity index of 0.8548. The genetic fingerprints of isolates recovered from recent outbreaks across different provinces were highly shared (100% similarity), suggesting clonal expansion within the production systems situated in the Mekong Delta region. Findings from this study also showed that E. ictaluri populations circulating this region have changed over time, as isolates recovered between 2001 and 2011 were genetically distinct from those recovered after 2017. Furthermore, isolates recovered after 2004 and 2010 lacked the virulence gene traD and a 5.8 Kb plasmid DNA fragment, respectively. Findings from this study illustrate the need for continued epidemiological monitoring of E. ictaluri to ensure prevention and treatment strategies in the Vietnamese striped catfish sector remain robust and effective.