Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

Resistance phenotype and virulence potential of Leclercia adecarboxylata strains isolated from different sources.

Introduction. Leclercia adecarboxylata is a member of Enterobacterales, often considered an opportunistic pathogen. Recent reports have highlighted L. adecarboxylata as an emerging pathogen harbouring virulence and resistance determinants.Gap statement. Little information exists on virulence and resistance determinants in L. adecarboxylata strains isolated from environmental, food, and clinical samples.Aim. To determine the presence of resistance and virulence determinants and plasmid features in L. adecarboxylata strains isolated from environmental, food, and clinical samples, as well as their phylogenetic relationship.Results. All strains tested showed resistance to ß-lactams and quinolones but were sensitive to aminoglycosides and nitrofurans. However, even though fosfomycin resistance is considered a characteristic trait of L. adecarboxylata, the resistance phenotype was only observed in 50 % of the strains; bla TEM was the most prevalent BLEE gene (70 %), while the quinolone qnrB gene was observed in 60 % of the strains. Virulence genes were differentially observed in the strains, with adhesion-related genes being the most abundant, followed by toxin genes. Finally, all strains carried one to seven plasmid bands ranging from 7 to 125 kbps and harboured several plasmid addiction systems, such as ParDE, VagCD, and CcdAB in 80 % of the strains.Conclusions. L. adecarboxylata is an important emerging pathogen that may harbour resistance and virulence genes. Additionally, it has mobilizable genetic elements that may contribute to the dissemination of genetic determinants to other bacterial genera.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran.

BACKGROUND: Tuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates. METHODS: Nineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP). RESULTS: All 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ. CONCLUSIONS: Our results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

A challenging case of carbapenem resistant Klebsiella pneumoniae-related pyogenic liver abscess with capsular polysaccharide hyperproduction: a case report.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, necessitating the administration of polymyxin E (colistin) as a last-line antibiotic. Meanwhile, the mortality rate associated with colistin-resistant K. pneumoniae infections is seriously increasing. On the other hand, importance of administration of carbapenems in promoting colistin resistance in K. pneumoniae is unknown. CASE PRESENTATION: We report a case of K. pneumoniae-related pyogenic liver abscess in which susceptible K. pneumoniae transformed into carbapenem- and colistin-resistant K. pneumoniae during treatment with imipenem. The case of pyogenic liver abscess was a 50-year-old man with diabetes and liver transplant who was admitted to Abu Ali Sina Hospital in Shiraz. The K. pneumoniae isolate responsible for community-acquired pyogenic liver abscess was isolated and identified. The K. pneumoniae isolate was sensitive to all tested antibiotics except ampicillin in the antimicrobial susceptibility test and was identified as a non-K1/K2 classical K. pneumoniae (cKp) strain. Multilocus sequence typing (MLST) identified the isolate as sequence type 54 (ST54). Based on the patient's request, he was discharged to continue treatment at another center. After two months, he was readmitted due to fever and progressive constitutional symptoms. During treatment with imipenem, the strain acquired blaOXA-48 and showed resistance to carbapenems and was identified as a multidrug resistant (MDR) strain. The minimum inhibitory concentration (MIC) test for colistin was performed by broth microdilution method and the strain was sensitive to colistin (MIC < 2 µg/mL). Meanwhile, on blood agar, the colonies had a sticky consistency and adhered to the culture medium (sticky mucoviscous colonies). Quantitative real-time PCR and biofilm formation assay revealed that the CRKP strain increased capsule wzi gene expression and produced slime in response to imipenem. Finally, K. pneumoniae-related pyogenic liver abscess with resistance to a wide range of antibiotics, including the last-line antibiotics colistin and tigecycline, led to sepsis and death. CONCLUSIONS: Based on this information, can we have a theoretical hypothesis that imipenem is a promoter of resistance to carbapenems and colistin in K. pneumoniae? This needs more attention.

Molecular mechanisms of tigecycline-resistance among Enterobacterales.

The global emergence of antimicrobial resistance to multiple antibiotics has recently become a significant concern. Gram-negative bacteria, known for their ability to acquire mobile genetic elements such as plasmids, represent one of the most hazardous microorganisms. This phenomenon poses a serious threat to public health. Notably, the significance of tigecycline, a member of the antibiotic group glycylcyclines and derivative of tetracyclines has increased. Tigecycline is one of the last-resort antimicrobial drugs used to treat complicated infections caused by multidrug-resistant (MDR) bacteria, extensively drug-resistant (XDR) bacteria or even pan-drug-resistant (PDR) bacteria. The primary mechanisms of tigecycline resistance include efflux pumps' overexpression, tet genes and outer membrane porins. Efflux pumps are crucial in conferring multi-drug resistance by expelling antibiotics (such as tigecycline by direct expelling) and decreasing their concentration to sub-toxic levels. This review discusses the problem of tigecycline resistance, and provides important information for understanding the existing molecular mechanisms of tigecycline resistance in Enterobacterales. The emergence and spread of pathogens resistant to last-resort therapeutic options stands as a major global healthcare concern, especially when microorganisms are already resistant to carbapenems and/or colistin.

High prevalence of multidrug-resistant Enterobacterales carrying extended-spectrum beta-lactamase and AmpC genes isolated from neonatal sepsis in Ahvaz, Iran.

OBJECTIVES: In the recent years, multidrug resistant (MDR) neonatal septicemia-causing Enterobacterales has been dramatically increased due to the extended-spectrum beta-lactamases (ESBLs) and AmpC enzymes. This study aimed to assess the antibiotic resistance pattern, prevalence of ESBLs/AmpC beta-lactamase genes, and Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) fingerprints in Enterobacterales isolated from neonatal sepsis. RESULTS: In total, 59 Enterobacterales isolates including 41 (69.5%) Enterobacter species, 15 (25.4%) Klebsiella pneumoniae and 3 (5.1%) Escherichia coli were isolated respectively. Resistance to ceftazidime and cefotaxime was seen in all of isolates. Furthermore, all of them were multidrug-resistant (resistant to three different antibiotic categories). The phenotypic tests showed that 100% of isolates were ESBL-positive. Moreover, AmpC production was observed in 84.7% (n = 50/59) of isolates. Among 59 ESBL-positive isolates, the highest percentage belonged to blaCTX-M-15 gene (66.1%) followed by blaCTX-M (45.8%), blaCTX-M-14 (30.5%), blaSHV (28.8%), and blaTEM (13.6%). The frequency of blaDHA, blaEBC, blaMOX and blaCIT genes were 24%, 24%, 4%, and 2% respectively. ERIC-PCR analysis revealed that Enterobacterales isolates were genetically diverse. The remarkable prevalence of MDR Enterobacterales isolates carrying ESBL and AmpC beta-lactamase genes emphasizes that efficient surveillance measures are essential to avoid the more expansion of drug resistance amongst isolates.

Antimicrobial resistance and carbapenemase dissemination in Pseudomonas aeruginosa isolates from Libyan hospitals: a call for surveillance and intervention.

Pseudomonas aeruginosa is a multidrug-resistant bacterium capable of forming biofilms. This study aimed to assess resistance of clinical isolates from Libyan hospitals to antipseudomonal antibiotics, the prevalence of selected extended-spectrum ß-lactamases and carbapenemase genes among these isolates, and the microorganisms' capacity for alginate and biofilm production. Forty-five isolates were collected from four hospitals in Benghazi and Derna, Libya. Antimicrobial susceptibility was determined using agar disc diffusion. The presence of resistance genes (blaCTXM, blaTEM, blaSHV-1, blaGES-1, blaKPC, and blaNDM) was screened using PCR. Biofilm formation was quantified via the crystal violet assay, while alginate production was measured spectrophotometrically. Resistance to antipseudomonal antibiotics ranged from 48.9% to 75.6%. The most prevalent resistance gene was blaNDM (26.7%), followed by blaGES-1 (17.8%). Moreover, all isolates demonstrated varying degrees of biofilm-forming ability and alginate production. No statistically significant correlation was found between biofilm formation and alginate production. The dissemination of resistant genes in P. aeruginosa, particularly carbapenemases, is of great concern. This issue is compounded by the bacteria's biofilm-forming capability. Urgent intervention and continuous surveillance are imperative to prevent further deterioration and the catastrophic spread of resistance among these formidable bacteria.

A sophisticated virulence repertoire and colistin resistance of Citrobacter freundii ST150 from a patient with sepsis admitted to ICU in a tertiary care hospital in Uganda, East Africa: Insight from genomic and molecular docking analyses.

Sepsis and multidrug resistance comprise a complex of factors attributable to mortality among intensive care unit (ICU) patients globally. Pathogens implicated in sepsis are diverse, and their virulence and drug resistance remain elusive. From a tertiary care hospital ICU in Uganda, we isolated a Citrobacter freundii strain RSM030 from a patient with sepsis and phenotypically tested it against a panel of 16 antibiotics including imipenem levofloxacin, cotrimoxazole and colistin, among others. We sequenced the organism's genome and integrated multilocus sequencing (MLST), PathogenFinder with Virulence Factor analyzer (VFanalyzer) to establish its pathogenic relevance. Thereafter, we combined antiSMASH and PRISM genome mining with molecular docking to predict biosynthetic gene clusters (BGCs), pathways, toxin structures and their potential targets in-silico. Finally, we coupled ResFinder with comprehensive antibiotic resistance database (CARD) to scrutinize the genomic antimicrobial resistance profile of the isolate. From PathogenFinder and MLST, this organism was confirmed to be a human pathogen (p = 0.843), sequence type (ST)150, whose virulence is determined by chromosomal type III secretion system (T3SS) (the injectosome) and plasmid-encoded type IV secretion system (T4SS), the enterobactin biosynthetic gene cluster and biofilm formation through the pgaABCD operon. Pathway and molecular docking analyses revealed that the shikimate pathway can generate a toxin targeting multiple host proteins including spectrin, detector of cytokinesis protein 2 (Dock2) and plasmalemma vesicle-associated protein (PLVAP), potentially distorting the host cell integrity. From phenotypic antibiotic testing, we found indeterminate results for amoxicillin/clavulanate and levofloxacin, with resistance to cotrimoxazole and colistin. Detailed genome analysis revealed chromosomal beta lactam resistance genes, i.e. blaCMY-79, blaCMY-116 and blaTEM-1B, along with multiple mutations of the lipopolysaccharide modifying operon genes PmrA/PmrB, pmrD, mgrA/mgrB and PhoP/PhoQ, conferring colistin resistance. From these findings, we infer that Citrobacter freundii strain RSM030 is implicated in sepsis and resistance to standard antibiotics, including colistin, the last resort.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation.

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-ß-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to ß-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

Emergence and genomic chion of Proteus mirabilis harboring blaNDM-1 in Korean companion dogs.

Proteus mirabilis is a commensal bacterium dwelling in the gastrointestinal (GI) tract of humans and animals. Although New Delhi metallo-ß-lactamase 1 (NDM-1) producing P. mirabilis is emerging as a threat, its epidemiology in our society remains largely unknown. LHPm1, the first P. mirabilis isolate harboring NDM-1, was detected from a companion dog that resides with a human owner. The whole-genome study revealed 20 different antimicrobial resistance (AMR) genes against various classes of antimicrobial agents, which corresponded to the MIC results. Genomic regions, including MDR genes, were identified with multiple variations and visualized in a comparative manner. In the whole-genome epidemiological analysis, multiple phylogroups were identified, revealing the genetic relationship of LHPm1 with other P. mirabilis strains carrying various AMR genes. These genetic findings offer comprehensive insights into NDM-1-producing P. mirabilis, underscoring the need for urgent control measures and surveillance programs using a "one health approach".

The recent rapid expansion of multidrug resistant Ural lineage Mycobacterium tuberculosis in Moldova.

The projected trajectory of multidrug resistant tuberculosis (MDR-TB) epidemics depends on the reproductive fitness of circulating strains of MDR M. tuberculosis (Mtb). Previous efforts to characterize the fitness of MDR Mtb have found that Mtb strains of the Beijing sublineage (Lineage 2.2.1) may be more prone to develop resistance and retain fitness in the presence of resistance-conferring mutations than other lineages. Using Mtb genome sequences from all culture-positive cases collected over two years in Moldova, we estimate the fitness of Ural (Lineage 4.2) and Beijing strains, the two lineages in which MDR is concentrated in the country. We estimate that the fitness of MDR Ural strains substantially exceeds that of other susceptible and MDR strains, and we identify several mutations specific to these MDR Ural strains. Our findings suggest that MDR Ural Mtb has been transmitting efficiently in Moldova and poses a substantial risk of spreading further in the region.

Inferring Mycobacterium Tuberculosis Drug Resistance and Transmission using Whole-genome Sequencing in a High TB-burden Setting in China.

Objective: China is among the 30 countries with a high burden of tuberculosis (TB) worldwide, and TB remains a public health concern. Kashgar Prefecture in the southern Xinjiang Autonomous Region is considered as one of the highest TB burden regions in China. However, molecular epidemiological studies of Kashgar are lacking. Methods: A population-based retrospective study was conducted using whole-genome sequencing (WGS) to determine the characteristics of drug resistance and the transmission patterns. Results: A total of 1,668 isolates collected in 2020 were classified into lineages 2 (46.0%), 3 (27.5%), and 4 (26.5%). The drug resistance rates revealed by WGS showed that the top three drugs in terms of the resistance rate were isoniazid (7.4%, 124/1,668), streptomycin (6.0%, 100/1,668), and rifampicin (3.3%, 55/1,668). The rate of rifampicin resistance was 1.8% (23/1,290) in the new cases and 9.4% (32/340) in the previously treated cases. Known resistance mutations were detected more frequently in lineage 2 strains than in lineage 3 or 4 strains, respectively: 18.6% vs. 8.7 or 9%, P < 0.001. The estimated proportion of recent transmissions was 25.9% (432/1,668). Multivariate logistic analyses indicated that sex, age, occupation, lineage, and drug resistance were the risk factors for recent transmission. Despite the low rate of drug resistance, drug-resistant strains had a higher risk of recent transmission than the susceptible strains (adjusted odds ratio, 1.414; 95% CI, 1.023-1.954; P = 0.036). Among all patients with drug-resistant tuberculosis (DR-TB), 78.4% (171/218) were attributed to the transmission of DR-TB strains. Conclusion: Our results suggest that drug-resistant strains are more transmissible than susceptible strains and that transmission is the major driving force of the current DR-TB epidemic in Kashgar.

[Genetic characterization and drug resistance analysis of Salmonella Kentucky ST314 in Shenzhen in 2010-2021].

OBJECTIVE: To understand the prevalence, genetic characteristics and drug resistance features of Salmonella Kentucky ST314 in Shenzhen. METHODS: Whole genome sequencing of 14 strains of Salmonella Kentucky ST314 collected from 2010-2021 by the Foodborne Disease Surveillance Network of Shenzhen Center for Disease Control and Prevention for phylogenetic evolutionary analysis, drug resistance gene and plasmid detection; drug susceptibility experiments were performed by micro-broth dilution method. RESULTS: A total of 57 strains of Salmonella Kentucky were collected from the foodborne disease surveillance network, 14 of which were ST314. The Shenzhen isolates were clustered with isolates from Southeast Asian countries such as Vietnam and Thailand on clade 314.2, and the single nucleotide polymorphism distance between local strains in Shenzhen was large, indicating dissemination. In this study, a total of 17 drug resistance genes/mutations in 9 categories were detected in the genome of Salmonella Kentucky ST314, carrying 3 extended spectrum beta-lactamases(ESBLs), including bla_(CTX-M-24)(14.3%, 2/14), bla_(CTX-M-55)(7.1%, 1/14), and bla_(CTX-M-130)(14.3%, 2/14), all located on plasmids. Regarding quinolone resistance factors, two plasmid-mediated quinolone resistance(PMQR) genes were identified in the genome: qnrB6(71.4%, 10/14) and aac(6')Ib-cr(78.6%, 11/14), a quinolone resistance quinolone resistance-determining regions(QRDR) mutation T57 S(100%, 14/14). The multi-drug resistance rate of Salmonella Kentucky ST314 in Shenzhen was 92.86%(13/14)with the highest rate of resistance to tetracycline and cotrimoxazole(100%, 14/14), followed by chloramphenicol(92.86%, 13/14), cefotaxime and ampicillin(78.57%, 11/14), ciprofloxacin and nalidixic acid(71.43%, 10/14), and ampicillin-sulbactam had the lowest resistance rate(21.43%, 3/14). CONCLUSION: ST314 is the second most prevalent ST type among Salmonella Kentucky in Shenzhen, mainly isolated from food, especially poultry; phylogenetic analysis suggests that ST314 is a disseminated infection and the genome shows a highly genetically conserved phenotype. Drug resistance of Salmonella Kentucky ST314 is very serious, especially QRDR mutation, PMQR gene co-mediated quinolone resistance and plasmid-mediated cephalosporin resistance are prominent and deserve extensive attention.

Deciphering the molecular and functional basis of TMexCD1: the plasmid-encoded efflux pump of resistance-nodulation-division superfamily.

Horizontal gene transfer has been demonstrated to be an important driver for the emergency of multidrug-resistant pathogens. Recently, a transferable gene cluster tmexCD1-toprJ1 of the resistance-nodulation-division (RND) superfamily was identified in the plasmids of animal-derived Klebsiella pneumoniae strains, with a higher efflux capacity for various drugs than the Escherichia coli AcrAB-TolC homolog system. In this study, we focused on the differences in the inner membrane pump of these two systems and identified some key residues that contribute to the robust efflux activity of the TMexCD1 system. With the aid of homologous modeling and molecular docking, eight residues from the proximal binding pocket (PBP) and nine from the distal binding pocket (DBP) were selected and subjected to site-directed mutagenesis. Several of them, such as S134, I139, D181, and A290, were shown to be important for substrate binding in the DBP region, and all residues in PBP and DBP showed certain substrate preferences. Apart from the conservative switch loop (L613-623TMexD1) previously identified in the E. coli AcrB (EcAcrB), a relatively unconservative loop (L665-675TMexD1) at the bottom of PBP was proposed as a critical element for the robust activity of TMexD1, due to variations at sites E669, G670, N673, and S674 compared to EcAcrAB, and the significantly altered efflux activity due to their mutations. The conservation and flexibility of these key factors can contribute to the evolution of the RND efflux pumps and thus serve as potential targets for developing inhibitors to block the widespread of the TMexCD1 system.

Multilocus sequence typing and antibiotic resistance of Aeromonas isolated from freshwater fish in Hebei Province.

Aeromonas spp. are the opportunistic pathogens that infect both aquatic and terrestrial homeotherms. They were commonly present in aquatic environments, including effluent, tap water, marine, river, and lake, where they are often isolated from aquatic animals, including fish, molluscs, and crustaceans. The Aeromonas infections can cause sepsis, ulcer, and other symptoms, resulting in the death of massive aquatic animals. Therefore, the prevention and control of Aeromonas is of great significance for the healthy development of aquaculture. In this study, we used modern molecular methods to enhance disease control of Aeromonas isolates from freshwater fish in Hebei Province. A total of 130 Aeromonas spp. isolates were isolated from freshwater fish farms in Hengshui, Handan, and Shijiazhuang and all 130 Aeromonas spp. isolates were sequenced for species identification. Of the 130 Aeromonas spp. isolates, 104 isolates were successfully sequenced, and BLAST analysis showed that Aeromonas veronii was predominant in freshwater fish farms in Hebei Province. In addition, 26 antibiotic resistance profiles were obtained from 102 fully cultured isolates among the 104 Aeromonas spp. isolates whose species was primarily identified, and 44 multidrug-resistant bacteria among the 102 isolates were identified using an antibiotic susceptibility test. Using the Multilocus Sequence Typing (MLST) method, 33 out of 44 multidrug-resistant isolates with 14 non-Aeromonas reference strains were selected for phylogenetic and MLST analysis, and all 33 multidrug-resistant isolates were A. veronii. A total of 30 new Sequence Types (STs) were obtained by comparing concatenated sequences (gyrB-groL-gltA-metG-ppsA-recA) on PubMLST website. Furthermore, recombination event analysis detected using RDP5 and ClonalFrameML software 42 and 49 recombination events, respectively, and 22 recombination events were validated by four or more algorithms. Since mutation and recombination events increase clonal diversity and single housekeeping gene sequence alignments are limited for identifying species, we propose the use of multiple concatenated sequence loci to increase discriminatory power. In addition, we propose that the MLST method is an appropriate technique to study and develop the resistance mechanisms of multidrug-resistant Aeromonas and to identify Aeromonas systematically in complex samples obtained from the environment.

Antimicrobial Resistance and the Genomic Epidemiology of Multidrug-Resistant Salmonella enterica serovar Enteritidis ST11 in China.

BACKGROUND: With the recent evolution of multidrug-resistant strains, the genetic characteristics of foodborne Salmonella enterica serovar Enteritidis and clinical isolates have changed. ST11 is now the most common genotype associated with S. Enteritidis isolates. METHODS: A total of 83 strains of S. Enteritidis were collected at the General Hospital of the People's Liberation Army. Of these, 37 were from aseptic sites in patients, 11 were from the feces of patients with diarrhea, and the remaining 35 were of chicken-origin. The minimum inhibitory concentration of S. Enteritidis was determined by the broth microdilution method. Genomic DNA was extracted using the QiAamp DNA Mini Kit, and whole-genome sequencing (WGS) was performed using an Illumina X-ten platform. Prokka was used for gene prediction and annotation, and bioinformatic analysis tools included Resfinder, ISFinder, Virulence Factor Database, and PlasmidFinder. IQ-TREE was used to build a maximum likelihood phylogenetic tree. The phylogenetic relationship and distribution of resistance genes was displayed using iTOL. Comparative population genomics was used to analyze the phenotypes and genetic characteristics of antibiotic resistance in clinical and chicken-origin isolates of S. Enteritidis. RESULTS: The chicken-origin S. Enteritidis isolates were more resistant to antibiotics than clinical isolates, and had a broader antibiotic resistance spectrum and higher antibiotic resistance rate. A higher prevalence of antibiotic-resistance genes was observed in chicken-origin S. Enteritidis compared to clinical isolates, along with distinct patterns in the contextual characteristics of these genes. Notably, genes such as blaCTX-M and dfrA17 were exclusive to plasmids in clinical S. Enteritidis, whereas in chicken-origin S. Enteritidis they were found in both plasmids and chromosomes. Additionally, floR was significantly more prevalent in chicken-origin isolates than in clinical isolates. Careful analysis revealed that the delayed isolation of chicken-origin S. Enteritidis contributes to accelerated gene evolution. Of note, certain resistance genes tend to integrate seamlessly and persist steadfastly within the chromosome, thereby expediting the evolution of resistance mechanisms against antibiotics. Our comparative analysis of virulence genes in S. Enteritidis strains from various sources found no substantial disparities in the distribution of other virulence factors. In summary, we propose that chicken-origin S. Enteritidis has the potential to cause clinical infections. Moreover, the ongoing evolution and dissemination of these drug-resistant genes poses a formidable challenge to clinical treatment. CONCLUSIONS: Constant vigilance is needed to monitor the dynamic patterns of drug resistance in S. Enteritidis strains sourced from diverse origins.

Characterization of the diversity of type IV secretion system-encoding plasmids in Acinetobacter.

The multi-drug resistant pathogen Acinetobacter baumannii has gained global attention as an important clinical challenge. Owing to its ability to survive on surfaces, its capacity for horizontal gene transfer, and its resistance to front-line antibiotics, A. baumannii has established itself as a successful pathogen. Bacterial conjugation is a central mechanism for pathogen evolution. The epidemic multidrug-resistant A. baumannii ACICU harbours a plasmid encoding a Type IV Secretion System (T4SS) with homology to the E. coli F-plasmid, and plasmids with homologous gene clusters have been identified in several A. baumannii sequence types. However the genetic and host strain diversity, global distribution, and functional ability of this group of plasmids is not fully understood. Using systematic analysis, we show that pACICU2 belongs to a group of almost 120 T4SS-encoding plasmids within four different species of Acinetobacter and one strain of Klebsiella pneumoniae from human and environmental origin, and globally distributed across 20 countries spanning 4 continents. Genetic diversity was observed both outside and within the T4SS-encoding cluster, and 47% of plasmids harboured resistance determinants, with two plasmids harbouring eleven. Conjugation studies with an extensively drug-resistant (XDR) strain showed that the XDR plasmid could be successfully transferred to a more divergent A. baumanii, and transconjugants exhibited the resistance phenotype of the plasmid. Collectively, this demonstrates that these T4SS-encoding plasmids are globally distributed and more widespread among Acinetobacter than previously thought, and that they represent an important potential reservoir for future clinical concern.

Prevalence and characteristics of ESBL-producing Escherichia coli in clinically healthy pigs: implications for antibiotic resistance spread in livestock.

AIM: This study aimed to compare and characterize the resistance profile and the presence of extended-spectrum beta-lactamase (ESBL) related genes in Escherichia coli isolated from healthy finishing pigs fed with or without antibiotics in their diets. METHODS AND RESULTS: A total of 27 ceftiofur-resistant E. coli isolates were obtained from 96 healthy pigs. The antibiotic resistance profile was tested, and all 27 isolates were classified as multidrug-resistant (MDR). A high proportion of isolates were resistant to cephalosporins, ampicillin, ciprofloxacin, and tetracyclines. The ESBL production was observed in 85% of isolates by double-disc synergy test. The MDR-E. coli isolates harbored ESBL genes, such as blaTEM, blaCTX-M-1, blaCTX-M-2, and blaCTX-M-8,25. In addition, other antibiotics resistance genes (ARGs) were also detected, such as sul2, ant(3″)-I, tetA, and mcr-1. The mobilization of the blaCTX-M gene was confirmed for nine E. coli isolates by conjugation assays. The presence of blaCTX-M on mobile genetic elements in these isolates was demonstrated by Southern blot hybridization, and the resistance to cephalosporins was confirmed in the transconjugants. Our results indicate the prevalence of CTX-M-producing E. coli strains harboring mobile genetic elements in the normal microbiota of healthy pigs. CONCLUSIONS: These findings highlight the significance of ESBL genes as a global health concern in livestock and the potential spread of antimicrobial resistance to other members of the gastrointestinal tract microbiota.