sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

Clinical and epidemiological characteristics of multi-drug resistant Enterobacterales isolated from King Fahad Hospital of the University, AlKhobar, Saudi Arabia.

Multi-drug resistant (MDR) Enterobacterales remain a major clinical problem. Infections caused by carbapenem-resistant strains are particularly difficult to treat. This study aimed to assess the clinical and epidemiological characteristics of MDR Enterobacterales isolates. A total of 154 non-repetitive clinical isolates, including Escherichia coli (n = 66), Klebsiella pneumoniae (n = 70), and other Enterobacterales (n = 18), were collected from the Diagnostic Microbiology Laboratory at King Fahad Hospital of the University. Most E. coli isolates were collected from urine specimens (n = 50, 75.8%) and resistance against the third and fourth-generation cephalosporins (ceftriaxone, ceftazidime, cefixime, and cefepime) and fluoroquinolones (ciprofloxacin and levofloxacin) was assessed. Clonal relatedness analysis using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR) revealed two clones (E. coli A and B), each comprising two strains. Most K. pneumoniae samples were collected from respiratory specimens (27.1%, 20 samples), and the strains showed overall resistance to most of the antimicrobials tested (54%‒100%). Moreover, clonal-relatedness analysis using ERIC-PCR revealed seven major clones of K. pneumoniae. These findings suggest nosocomial transmission among some identical strains and emphasize the importance of strict compliance with infection prevention and control policies and regulations. Environmental reservoirs could facilitate this indirect transmission, which needs to be investigated.

Performance comparison of BD Phoenix CPO detect panel with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Klebsiella pneumoniae isolates.

BACKGROUND: We aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L. RESULTS: According to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively. CONCLUSION: While the CPO panel's phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.

Implications of deduplication on the detection rates of multidrug-resistant organism (MDRO) in various specimens: insights from the hospital infection surveillance program.

BACKGROUND: Currently, different guidelines recommend using different methods to determine whether deduplication is necessary when determining the detection rates of multidrug-resistant organisms (MDROs). However, few studies have investigated the effect of deduplication on MDRO monitoring data. In this study, we aimed to investigate the influence of deduplication on the detection rates of MDROs in different specimens to assess its impact on infection surveillance outcomes. METHODS: Samples were collected from hospitalized patients admitted between January 2022 and December 2022; four types of specimens were collected from key monitored MDROs, including sputum samples, urine samples, blood samples, and bronchoalveolar lavage fluid (BALF) samples. In this study, we compared and analysed the detection rates of carbapenem-resistant Klebsiella pneumoniae (CRKP), carbapenem-resistant Escherichia coli (CRECO), carbapenem-resistant Acinetobacter baumannii (CRAB), carbapenem-resistant Pseudomonas aeruginosa (CRPA), and methicillin-resistant Staphylococcus aureus (MRSA) under two conditions: with and without deduplication. RESULTS: When all specimens were included, the detection rates of CRKP, CRAB, CRPA, and MRSA without deduplication (33.52%, 77.24%, 44.56%, and 56.58%, respectively) were significantly greater than those with deduplication (24.78%, 66.25%, 36.24%, and 50.83%, respectively) (all P < 0.05). The detection rates in sputum samples were significantly different between samples without duplication (28.39%, 76.19%, 46.95%, and 70.43%) and those with deduplication (19.99%, 63.00%, 38.05%, and 64.50%) (all P < 0.05). When deduplication was not performed, the rate of detection of CRKP in urine samples reached 30.05%, surpassing the rate observed with deduplication (21.56%) (P < 0.05). In BALF specimens, the detection rates of CRKP and CRPA without deduplication (39.78% and 53.23%, respectively) were greater than those with deduplication (31.62% and 42.20%, respectively) (P < 0.05). In blood samples, deduplication did not have a significant impact on the detection rates of MDROs. CONCLUSION: Deduplication had a significant effect on the detection rates of MDROs in sputum, urine, and BALF samples. Based on these data, we call for the Infection Prevention and Control Organization to align its analysis rules with those of the Bacterial Resistance Surveillance Organization when monitoring MDRO detection rates.

Assessment of three antibiotic combination regimens against Gram-negative bacteria causing neonatal sepsis in low- and middle-income countries.

Gram-negative bacteria (GNB) are a major cause of neonatal sepsis in low- and middle-income countries (LMICs). Although the World Health Organization (WHO) reports that over 80% of these sepsis deaths could be prevented through improved treatment, the efficacy of the currently recommended first- and second-line treatment regimens for this condition is increasingly affected by high rates of drug resistance. Here we assess three well known antibiotics, fosfomycin, flomoxef and amikacin, in combination as potential antibiotic treatment regimens by investigating the drug resistance and genetic profiles of commonly isolated GNB causing neonatal sepsis in LMICs. The five most prevalent bacterial isolates in the NeoOBS study (NCT03721302) are Klebsiella pneumoniae, Acinetobacter baumannii, E. coli, Serratia marcescens and Enterobacter cloacae complex. Among these isolates, high levels of ESBL and carbapenemase encoding genes are detected along with resistance to ampicillin, gentamicin and cefotaxime, the current WHO recommended empiric regimens. The three new combinations show excellent in vitro activity against ESBL-producing K. pneumoniae and E. coli isolates. Our data should further inform and support the clinical evaluation of these three antibiotic combinations for the treatment of neonatal sepsis in areas with high rates of multidrug-resistant Gram-negative bacteria.

Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance.

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae.

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam.

Hypervirulent Klebsiella pneumoniae (hvKp) is a significant cause of severe invasive infections in Vietnam, yet data on its epidemiology, population structure and dynamics are scarce. We screened hvKp isolates from patients with bloodstream infections (BSIs) at a tertiary infectious diseases hospital in Vietnam and healthy individuals, followed by whole genome sequencing and plasmid analysis. Among 700 BSI-causing Kp strains, 100 (14.3%) were hvKp. Thirteen hvKp isolates were identified from 350 rectal swabs of healthy adults; none from 500 rectal swabs of healthy children. The hvKp isolates were genetically diverse, encompassing 17 sequence types (STs), predominantly ST23, ST86 and ST65. Among the 113 hvKp isolates, 14 (12.6%) carried at least one antimicrobial resistance (AMR) gene, largely mediated by IncFII, IncR, and IncA/C plasmids. Notably, the acquisition of AMR conjugative plasmids facilitated horizontal transfer of the non-conjugative virulence plasmid between K. pneumoniae strains. Phylogenetic analysis demonstrated hvKp isolates from BSIs and human carriage clustered together, suggesting a significant role of intestinal carriage in hvKp transmission. Enhanced surveillance is crucial to understand the factors driving intestinal carriage and hvKp transmission dynamics for informing preventive measures. Furthermore, we advocate the clinical use of our molecular assay for diagnosing hvKp infections to guide effective management.

[Effect of intestinal nitrate on growth of Klebsiella pneumoniae and its regulatory mechanism].

OBJECTIVE: To explore the effect of intestinal nitrates on the growth of Klebsiella pneumoniae and its regulatory mechanisms. METHODS: K. pneumoniae strains with nitrate reductase narG and narZ single or double gene knockout or with NarXL gene knockout were constructed and observed for both aerobic and anaerobic growth in the presence of KNO3 using an automated bacterial growth analyzer and a spectrophotometer, respectively. The mRNA expressions of narG and narZ in K. pneumoniae in anaerobic cultures in the presence of KNO3 and the effect of the binary regulatory system NarXL on their expresisons were detected using qRT-PCR. Electrophoretic mobility shift assays (EMSA) and MST analysis were performed to explore the specific regulatory mechanisms of NarXL in sensing and utilizing nitrates. Competitive experiments were conducted to examine anaerobic growth advantages of narG and narZ gene knockout strains of K. pneumoniae in the presence of KNO3. RESULTS: The presence of KNO3 in anaerobic conditions, but not in aerobic conditions, promoted bacterial growth more effectively in the wild-type K. pneumoniae strain than in the narXL gene knockout strain. In anaerobic conditions, the narXL gene knockout strain showed significantly lowered mRNA expressions of narG and narZ (P < 0.0001). EMSA and MST experiments demonstrated that the NarXL regulator could directly bind to narG and narZ promoter regions. The wild-type K. pneumoniae strain in anaerobic cultures showed significantly increased expressions of narG and narZ mRNAs in the presence of KNO3 (P < 0.01), and narG gene knockout resulted in significantly attenuated anaerobic growth and competitive growth abilities of K. pneumoniae in the presence of KNO3 (P < 0.01). CONCLUSION: The binary regulatory system NarXL of K. pneumoniae can sense changes in intestinal nitrate concentration and directly regulate the expression of nitrate reductase genes narG and narZ to promote bacterial growth.

[Construction and characterization of a modA gene mutant strain of Klebsiella pneumoniae].

OBJECTIVE: To construct a mutant strain of Klebsiella pneumoniae NTUH- K2044 with modA gene deletion and its complementary strain and explore the role of modA gene in modulating anaerobic nitrate respiratory growth and phenotypes of K. pneumoniae. METHODS: The modA deletion mutant K. pneumoniae strain was constructed by homologous recombination using the suicide vector pKO3-Km. To obtain the complementary strain C-modA, the whole sequence fragment containing the promoter, open reading frame and terminator regions of modA was cloned into pGEM-T-easy and electrically transformed into the modA deletion mutant. The NTUH-K2044 wild-type strain, modA gene deletion mutant and complementary strain were compared by measuring in vitro anaerobic nitrate respiration growth, competitiveness index, biofilm quantification, mucoviscosity assay and morphological measurement using Image J. RESULTS: The modA deletion mutant strain ΔmodA and the complementary strain C-modA were successfully constructed. The modA gene knockout strain showed inhibited anaerobic nitrate respiratory growth compared with the wild- type and C-modA strains with significantly weakened competitiveness, reduced capacity of biofilm synthesis during anaerobiosis, and lowered mucoviscosity under anaerobic conditions. The ΔmodA strain showed a spherical morphology in anaerobic conditions as compared with the normal short rod-like morphology of K. pneumoniae, with also distinctly shorter length than the wild-type and C-modA strains. CONCLUSION: The molybdate transport system encoding gene modA is associated with the pathogenic capacity of K. pneumoniae by modulating its anaerobic nitrate respiration, competitiveness, biofilm formation, hypermucoviscous phenotype and morphology.

Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing.

Whole-genome reconstruction of bacterial pathogens has become an important tool for tracking transmission and antimicrobial resistance gene spread, but highly accurate and complete assemblies have largely only historically been achievable using hybrid long- and short-read sequencing. We previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcell/chemistry produced improved assemblies over the R9.4.1/kit10 combination, however long-read only assemblies contained more errors compared to Illumina-ONT hybrid assemblies. ONT have since released an R10.4.1/kit14 flowcell/chemistry upgrade and recommended the use of Bovine Serum Albumin (BSA) during library preparation, both of which reportedly increase accuracy and yield. They have also released updated basecallers trained using native bacterial DNA containing methylation sites intended to fix systematic basecalling errors, including common adenosine (A) to guanine (G) and cytosine (C) to thymine (T) substitutions. To evaluate these improvements, we successfully sequenced four bacterial reference strains, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, and nine genetically diverse E. coli bloodstream infection-associated isolates from different phylogroups and sequence types, both with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. In this small evaluation of 13 isolates we found that nanopore long-read-only R10.4.1/kit 14 assemblies with updated basecallers trained using bacterial methylated DNA produce accurate assemblies with ≥40×depth, sufficient to be cost-effective compared with hybrid ONT/Illumina sequencing in our setting.

Emergence of NDM-producing Enterobacterales infections in companion animals from Argentina.

Antimicrobial resistance is considered one of the most critical threat for both human and animal health. Recently, reports of infection or colonization by carbapenemase-producing Enterobacterales in companion animals had been described. This study report the first molecular characterization of NDM-producing Enterobacterales causing infections in companion animals from Argentina. Nineteen out of 3662 Enterobacterales isolates analyzed between October 2021 and July 2022 were resistant to carbapenemes by VITEK2C and disk diffusion method, and suspected to be carbapenemase-producers. Ten isolates were recovered from canine and nine from feline animals. Isolates were identified as K. pneumoniae (n = 9), E. coli (n = 6) and E. cloacae complex (n = 4), and all of them presented positive synergy among EDTA and carbapenems disks, mCIM/eCIM indicative of metallo-carbapenemase production and were also positive by PCR for blaNDM gene. NDM variants were determined by Sanger sequencing method. All 19 isolates were resistant to ß-lactams and aminoglycosides but remained susceptible to colistin (100%), tigecycline (95%), fosfomycin (84%), nitrofurantoin (63%), minocycline (58%), chloramphenicol (42%), doxycycline (21%), enrofloxacin (5%), ciprofloxacin (5%) and trimethoprim/sulfamethoxazole (5%). Almost all isolates (17/19) co-harbored blaCTX-M plus blaCMY, one harbored blaCTX-M alone and the remaining blaCMY. E. coli and E. cloacae complex isolates harbored blaCTX-M-1/15 or blaCTX-M-2 groups, while all K. pneumoniae harbored only blaCTX-M-1/15 genes. All E. coli and E. cloacae complex isolates harbored blaNDM-1, while in K. pneumoniae blaNDM-1 (n = 6), blaNDM-5 (n = 2), and blaNDM-1 plus blaNDM-5 (n = 1) were confirmed. MLST analysis revealed the following sequence types by species, K. pneumoniae: ST15 (n = 5), ST273 (n = 2), ST11, and ST29; E. coli: ST162 (n = 3), ST457, ST224, and ST1196; E. cloacae complex: ST171, ST286, ST544 and ST61. To the best of our knowledge, this is the first description of NDM-producing E. cloacae complex isolates recovered from cats. Even though different species and clones were observed, it is remarkable the finding of some major clones among K. pneumoniae and E. coli, as well as the circulation of NDM as the main carbapenemase. Surveillance in companion pets is needed to detect the spread of carbapenem-resistant Enterobacterales and to alert about the dissemination of these pathogens among pets and humans.

Ethanolamine metabolism through two genetically distinct loci enables Klebsiella pneumoniae to bypass nutritional competition in the gut.

Successful microbial colonization of the gastrointestinal (GI) tract hinges on an organism's ability to overcome the intense competition for nutrients in the gut between the host and the resident gut microbiome. Enteric pathogens can exploit ethanolamine (EA) in the gut to bypass nutrient competition. However, Klebsiella pneumoniae (K. pneumoniae) is an asymptomatic gut colonizer and, unlike well-studied enteric pathogens, harbors two genetically distinct ethanolamine utilization (eut) loci. Our investigation uncovered unique roles for each eut locus depending on EA utilization as a carbon or nitrogen source. Murine gut colonization studies demonstrated the necessity of both eut loci in the presence of intact gut microbiota for robust GI colonization by K. pneumoniae. Additionally, while some Escherichia coli gut isolates could metabolize EA, other commensals were incapable, suggesting that EA metabolism likely provides K. pneumoniae a selective advantage in gut colonization. Molecular and bioinformatic analyses unveiled the conservation of two eut loci among K. pneumoniae and a subset of the related taxa in the K. pneumoniae species complex, with the NtrC-RpoN regulatory cascade playing a pivotal role in regulation. These findings identify EA metabolism as a critical driver of K. pneumoniae niche establishment in the gut and propose microbial metabolism as a potential therapeutic avenue to combat K. pneumoniae infections.

Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Generation and maintenance of the circularized multimeric IS26-associated translocatable unit encoding multidrug resistance.

In gram-negative bacteria, IS26 often exists in multidrug resistance (MDR) regions, forming a pseudocompound transposon (PCTn) that can be tandemly amplified. It also generates a circular intermediate called the "translocatable unit (TU)", but the TU has been detected only by PCR. Here, we demonstrate that in a Klebsiella pneumoniae MDR clone, mono- and multimeric forms of the TU were generated from the PCTn in a preexisting MDR plasmid where the inserted form of the TU was also tandemly amplified. The two modes of amplification were reproduced by culturing the original clone under antimicrobial selection pressure, and the amplified state was maintained in the absence of antibiotics. Mono- and multimeric forms of the circularized TU were generated in a RecA-dependent manner from the tandemly amplified TU, which can be generated in RecA-dependent and independent manners. These findings provide novel insights into the dynamic processes of genome amplification in bacteria.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Molecular epidemiology of carbapenem-resistant gram-negative bacilli in Ecuador.

INTRODUCTION: Carbapenem-resistant gram-negative bacilli are a worldwide concern because of high morbidity and mortality rates. Additionally, the increasing prevalence of these bacteria is dangerous. To investigate the extent of antimicrobial resistance and prioritize the utility of novel drugs, we evaluated the molecular characteristics and antimicrobial susceptibility profiles of carbapenem-resistant Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii in Ecuador in 2022. METHODS: Ninety-five clinical isolates of carbapenem non-susceptible gram-negative bacilli were collected from six hospitals in Ecuador. Carbapenem resistance was confirmed with meropenem disk diffusion assays following Clinical Laboratory Standard Institute guidelines. Carbapenemase production was tested using a modified carbapenemase inactivation method. Antimicrobial susceptibility was tested with a disk diffusion assay, the Vitek 2 System, and gradient diffusion strips. Broth microdilution assays were used to assess colistin susceptibility. All the isolates were screened for the blaKPC, blaNDM, blaOXA-48, blaVIM and blaIMP genes. In addition, A. baumannii isolates were screened for the blaOXA-23, blaOXA-58 and blaOXA-24/40 genes. RESULTS: Carbapenemase production was observed in 96.84% of the isolates. The blaKPC, blaNDM and blaOXA-48 genes were detected in Enterobacterales, with blaKPC being predominant. The blaVIM gene was detected in P. aeruginosa, and blaOXA-24/40 predominated in A. baumannii. Most of the isolates showed co-resistance to aminoglycosides, fluoroquinolones, and trimethoprim/sulfamethoxazole. Both ceftazidime/avibactam and meropenem/vaborbactam were active against carbapenem-resistant gram-negative bacilli that produce serin-carbapenemases. CONCLUSION: The epidemiology of carbapenem resistance in Ecuador is dominated by carbapenemase-producing K. pneumoniae harbouring blaKPC. Extensively drug resistant (XDR) P. aeruginosa and A. baumannii were identified, and their identification revealed the urgent need to implement strategies to reduce the dissemination of these strains.