Clonal spread of carbapenemase-producing Enterobacteriaceae in a region, China.

BACKGROUND: The increasing number of carbapenemase-producing Enterobacterales (CPE) has become a serious problem globally. This study aimed to elucidate their geographically epidemiological characteristics. METHODS: Resistance genes were identified by polymerase chain reaction (PCR) and sequencing. Bacterial genotyping was studied using multilocus sequence typing (MLST) and wzi typing. The transferability of carbapenemase genes was determined by a broth mating method. The relationships between the rates of antimicrobial consumption and the prevalence of CRE were performed by Pearson's or Spearman's correlation analyses. RESULTS: A total of 930 phenotypically confirmed carbapenem-resistant Enterobacterales (CRE) isolates collected from 19 hospitals were genotypically characterized. K. pneumoniae (KP) and E. coli isolates were 785 (85.14%) and 96 (10.41%) among 922 CPE isolates. Two major carbapenemase genes blaKPC-2 and blaNDM in CPE isolates accounted for 84.6% (n = 780) and 13.77% (n = 127). ST11 comprised 86.83% (633/729) of KPC-2 KP isolates. Different combinations of extended spectrum-ß-lactamase (ESBL) genes of blaSHV, blaCTX, and blaTEM were found in KPC-2 producing KP isolates, and blaCTM-M-14/15, blaSHV-11/12 and blaTEM-1 were common ESBL genotypes. The wzi typing method could further subdivide ST11 KP group into at least five subgroups, among which wzi209 (69.83%, 442/633) was the most frequently isolated, followed by wzi141 (25.28%, 160/633). Conjugation assays showed that high conjugation rates were observed in CPE (15.24%, 32/210) for NDM plasmids, but relatively low (8.1%, 17/210) for KPC-2 plasmids. Different STs, different wzis and temperature could influence plasmid conjugation efficiency. No associations between the rates of antibiotics consumption and CPE prevalence were observed. The number of intra-hospital and inter-hospital transfers of CPE patients increased gradually from 18 (17.82%, 101) and 12 (11.88%, 101) in 2015 to 63 (30.73%, 205) and 51 (24.88%, 205) in 2018 (p = 0.016 and p = 0.008), respectively. Evidence-based measures could effectively reduce the prevalence of ST11-wzi209 clone but failed to control the dissemination of ST11-wzi141 KP clone. CONCLUSIONS: Clonal spread of CPE, especially KPC-2 ST11 KP was the key factor contributing to the CPE increase in the region. Continued vigilance for the importations should be maintained. Coordinated regional interventions are urgently needed to reduce CPE threat.

Epidemiological, Virulence, and Antibiotic Resistance Analysis of Klebsiella pneumoniae, a Major Source of Threat to Livestock and Poultry in Some Regions of Xinjiang, China.

Klebsiella pneumoniae (K. pneumoniae) is recognized as a zoonotic pathogen with an increasing threat to livestock and poultry. However, research on K. pneumoniae of animal origin remains limited. To address the gap, a comprehensive investigation was carried out by collecting a total of 311 samples from the farms of four animal species (dairy cow, chicken, sheep, and pig) in selected areas of Xinjiang, China. Isolates were identified by khe gene amplification and 16S rRNA gene sequencing. Genotyping of K. pneumonia isolates was performed using wzi typing and multilocus sequence typing (MLST). PCR was employed to identify virulence and resistance genes. An antibiotic susceptibility test was conducted using the Kirby-Bauer method. The findings revealed an isolation of 62 K. pneumoniae strains, with an average isolation rate of 19.94%, with the highest proportion originating from cattle sources (33.33%). Over 85.00% of these isolates harbored six virulence genes (wabG, uge, fimH, markD, entB, and ureA); while more than 75.00% of isolates possessed four resistance genes (blaTEM, blaSHV, oqxA, and gyrA). All isolates exhibited complete resistance to ampicillin and demonstrated substantial resistance to sulfisoxazole, amoxicillin/clavulanic acid, and enrofloxacin, with an antibiotic resistance rate of more than 50%. Furthermore, 48.39% (30/62) of isolates were classified as multidrug-resistant (MDR) strains, with a significantly higher isolation rate observed in the swine farms (66.67%) compared to other farms. Genetic characterization revealed the classification of the 62 isolates into 30 distinct wzi allele types or 35 different sequence types (STs). Notably, we identified K. pneumoniae strains of dairy and swine origin belonging to the same ST42 and wzi33-KL64 types, as well as strains of dairy and chicken origin belonging to the same wzi31-KL31-K31 type. These findings emphasize the widespread occurrence of drug-resistant K. pneumoniae across diverse animal sources in Xinjiang, underscoring the high prevalence of multidrug resistance. Additionally, our results suggest the potential for animal-to-animal transmission of K. pneumoniae and there was a correlation between virulence genes and antibiotic resistance genes. Moreover, the current study provides valuable data on the prevalence, antibiotic resistance, and genetic diversity of K. pneumoniae originating from diverse animal sources in Xinjiang, China.

Screening of mcr-1 among Gram-Negative Bacteria from Different Clinical Samples from ICU Patients in Alexandria, Egypt: One-Year Study.

Antimicrobial resistance represents a global dilemma. Our present study aimed to investigate the presence of mcr-1 among different Gram-negative bacteria including Enterobacteriaceae (except intrinsically resistant to colistin) and Pseudomonas aeruginosa. Gram-negative bacterial isolates were collected from different ICUs in several Alexandria hospitals from June 2019 to June 2020. The identification of these Gram-negative isolates was made using the VITEK-2® system (BioMérieux, France). SYBR Green-based PCR was used to screen for the presence of mcr-1 using a positive control that we amplified and sequenced earlier in our pilot study. All isolates were screened for the presence of mcr-1 regardless of their colistin susceptibility. Isolates that harbored mcr-1 were tested for colistin susceptibility and for the presence of some beta-lactamase genes. Klebsiella pneumoniae isolates harboring mcr-1 were capsule typed using the wzi sequence analysis. Four hundred eighty isolates were included in this study. Only six isolates harbored mcr-1.1. Of these, four were resistant to colistin, while two (K. pneumoniae and P. aeruginosa) were susceptible to colistin. Five of the six isolates were resistant to carbapenems. They harbored bla OXA-48, and three of them co-harbored bla NDM-1. K-58 was the most often found among our K. pneumoniae harboring mcr-1.1. To our knowledge, this is the first time to report colistin susceptible P. aeruginosa and K. pneumoniae harboring the mcr-1.1 gene in Egypt. Further studies are needed to investigate the presence of the mcr genes among colistin susceptible isolates to shed more light on its significance as a potential threat.

Epidemiology, Environmental Risks, Virulence, and Resistance Determinants of Klebsiella pneumoniae From Dairy Cows in Hubei, China.

Klebsiella pneumoniae (K. pneumoniae) is an opportunistic pathogen, which causes serious infections in humans and animals. To investigate the antimicrobial resistance pattern and virulence profile of K. pneumoniae, a total of 887 samples were collected from both the healthy and mastitis cows and the bedding, feed, feces, air, drinking water, spraying water, washing water, and milk cup swabs from five dairy farms in Hubei, China, during 2019 and 2020. K. pneumoniae was isolated and identified using PCR of the khe and 16S rDNA sequencing. A genotypic characterization was performed for K. pneumoniae isolates using wzi typing and multilocus sequence typing (MLST). Antimicrobial resistances were confirmed using broth microdilution against 17 antimicrobial agents and resistance and virulence genes were determined by PCR. The prevalence of K. pneumoniae was 26.94% (239/887) distributed in 101 wzi allele types (199/239, 83.26%) and 100 sequence types (STs) (209/239, 87.45%), including 5 new wzi allele type and 25 new STs. Phylogenetic analysis showed that K. pneumoniae isolated from milk, nipple swab, feed, and feces is classified in the same clone complex. By comparing with the PubMLST database, at least 67 STs have the risk of spreading in different species and regions. Interestingly, 60 STs have been isolated from humans. The isolates were highly sensitive to meropenem and colistin, but resistant to ampicillin (100%), sulfisoxazole (94.56%), cephalothin (47.28%), streptomycin (30.13%), and so on. Noteworthy, multidrug-resistant (MDR) rate was found to be 43.93% in this study. By PCR, 30 of 68 antimicrobial resistance (AMR) genes were identified; the prevalence rate of blaTEM, blaSHV, strA, strB, aadA1, and aac(6')-Ib-cr was more than 50%. Eleven CTX-M-producing K. pneumoniae were found. The detection rate of fimH, mrkD, uge, wabG, entB, iutA, iroN, and ureA was over 85%. This study reinforces the epidemiological importance of K. pneumoniae in food-producing animals in Hubei. The emergence and spread of environmental MDR K. pneumoniae may pose a potential threat to food safety and public health.

Cooccurrence of Antibiotic Resistance and Hypervirulence in High-Risk Carbapenem-Resistant K14.K64 and Wzi209 Klebsiella pneumoniae Strains Driven by Plasmids and Their Derivatives.

Emerging hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is a severe public health problem worldwide. To assess the cooccurrence of CRKP and hv-CRKP, a total of 1,181 CRKP isolates were collected from 2009 to 2018, covering their initial occurrence to outbreaks. Overall, two major capsular serotypes, namely, wzi209-CRKP and K14.K64-CRKP, were identified as being prevalent in pediatric and adult patients, respectively. Most isolates carried blaKPC, and the blaKPC-carrying hybrid plasmid IncFII-IncR, which was stable and transferable, was identified. The conjugation region (traN/traC) of IncFII-IncR was found to be variable, and the genes were used as markers to identify the transmission of strains among patient groups in this study. Notably, hv-CRKP was characterized by screening for four virulence genes (rmpA, iroN, terW, and rmpA2) in all 977 blaKPC-carrying K14.K64-CRKP and wzi209-CRKP strains. Two virulence types, namely, rmpA/iroN/terW/rmpA2 positive and terW/rmpA2 positive, were found. The corresponding virulence plasmids Vir1, i.e., nonconjugative IncFIB(k)-IncHI1B, and Vir2, i.e., conjugative antibiotic-resistant IncFIB-IncHI1B, were further characterized. Both Vir1 and Vir2 were stable, and the transferability of Vir2 was significantly higher than that of IncFII-IncR. However, none of the Vir1- or Vir2-carrying strains exhibited the hypervirulent phenotype. Meanwhile, hv-CRKP (terW/rmpA2 positive) was found in late 2018 among wzi209-CRKP strains. The corresponding Vir2-related fragment was characterized as chromosomally integrated, which dramatically enhanced the virulence of wzi209-CRKP. Transmission of hv-CRKP among patient groups was also confirmed according to virulence elements. Taken together, CRKP and hv-CRKP occurred on a large scale. Plasmids and their derivatives played an important role on this process. Surveillance and intervention of hv-CRKP are urgently needed. IMPORTANCE Currently, an increasing number of hv-CRKP strains have been reported and pose a substantial threat to public health worldwide, because these strains are considered to be simultaneously hypervirulent, carbapenem resistant, and transmissible. In this study, we provided a complete transition process of CRKP and hv-CRKP from their early emergence to outbreak in 10 years. We identified two epidemic groups, K14.K64 (wzi64)-CRKP and wzi209-CRKP, in adult and pediatric patients, respectively. K14.K64 (wzi64)-CRKP was widely present, while wzi209-CRKP was rarely reported as an epidemic type. We discovered a large scale of hv-CRKP transmission from CRKP and determined the importance of antibiotic resistance and virulence plasmids and their derivatives for the transition of CRKP and hv-CRKP. Two virulence plasmids coexist in out hospital, but neither of them enhanced virulence. Notably, we found a newly emerged type of CRKP, hypervirulent wzi209-CRKP, which had dramatically enhanced virulence, making it a great threat to human health.

The Molecular Epidemiology of Prevalent Klebsiella pneumoniae Strains and Humoral Antibody Responses against Carbapenem-Resistant K. pneumoniae Infections among Pediatric Patients in Shanghai.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has caused wide dissemination among pediatric patients globally and thus has aroused public concern. Here, we investigated the clinical epidemiological characteristics of 140 nonreplicate clinical K. pneumoniae strains isolated from pediatric patients between January and December 2021. Of all isolates, 16.43% (23 of 140) were CRKP strains, which predominantly contained KPC carbapenemase. wzi sequencing demonstrated that KL47 (65.22%, 15 of 23) was the most frequent capsular type, followed by KL64 (17.39%, 4 of 23). A total of 23 CRKP strains were classified into three different O-genotypes, including OL101 (65.22%, 15 of 23), O1 (26.09%, 6 of 23), and O3 (8.7%, 2 of 23). Interestingly, KL47 strains were strongly associated with OL101, while KL64 strains were all linked with O1. Some capsule-deficient strains were identified by serological typing, phage-typing, depolymerase-typing, and uronic acid assay. In this study, compared with healthy children, higher titers of anti-capsular polysaccharides (CPS) IgG were first detected in the sera of K47 and K64 K. pneumoniae-infected children, which had the effective bactericidal activity against corresponding serotype K. pneumoniae strains. These findings will facilitate the development of novel therapeutic and vaccine strategies against K. pneumoniae infection in children. IMPORTANCE The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains resistant to numerous antibiotics and the limited therapeutic options available have become an urgent health threat to the immunocompromised pediatric population. Vaccines and antibodies, especially those targeting capsular polysaccharides, may be novel and effective prevention and treatment options. Thus, it is important to understand the spread of CRKP in pediatric populations. This research presents OL101:KL47 and O1:KL64 as the predominant combinations among CRKP strains in children in Shanghai, China. The primary carbapenemase gene is KPC in CRKP strains. Additionally, this study found elevated levels of anti-CPS IgG against K47 and K64 K. pneumoniae strains in pediatric patients for the first time. The significant bactericidal activity of these anti-CPS IgGs was confirmed.

Serum Antibody Responses against Carbapenem-Resistant Klebsiella pneumoniae in Infected Patients.

Capsular polysaccharide (CPS) heterogeneity within carbapenem-resistant Klebsiella pneumoniae (CR-Kp) strain sequence type 258 (ST258) must be considered when developing CPS-based vaccines. Here, we sought to characterize CPS-specific antibody responses elicited by CR-Kp-infected patients. Plasma and bacterial isolates were collected from 33 hospital patients with positive CR-Kp cultures. Isolate capsules were typed by wzi sequencing. Reactivity and measures of efficacy of patient antibodies were studied against 3 prevalent CR-Kp CPS types (wzi29, wzi154, and wzi50). High IgG titers against wzi154 and wzi50 CPS were documented in 79% of infected patients. Patient-derived (PD) IgGs agglutinated CR-Kp and limited growth better than naive IgG and promoted phagocytosis of strains across the serotype isolated from their donors. Additionally, poly-IgG from wzi50 and wzi154 patients promoted phagocytosis of nonconcordant CR-Kp serotypes. Such effects were lost when poly-IgG was depleted of CPS-specific IgG. Additionally, mice infected with wzi50, wzi154, and wzi29 CR-Kp strains preopsonized with wzi50 patient-derived IgG exhibited lower lung CFU than controls. Depletion of wzi50 antibodies (Abs) reversed this effect in wzi50 and wzi154 infections, whereas wzi154 Ab depletion reduced poly-IgG efficacy against wzi29 CR-Kp We are the first to report cross-reactive properties of CPS-specific Abs from CR-Kp patients through both in vitro and in vivo models.IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is a rapidly emerging public health threat that can cause fatal infections in up to 50% of affected patients. Due to its resistance to nearly all antimicrobials, development of alternate therapies like antibodies and vaccines is urgently needed. Capsular polysaccharides constitute important targets, as they are crucial for Klebsiella pneumoniae pathogenesis. Capsular polysaccharides are very diverse and, therefore, studying the host's capsule-type specific antibodies is crucial to develop effective anti-CPS immunotherapies. In this study, we are the first to characterize humoral responses in infected patients against carbapenem-resistant Klebsiella pneumoniae expressing different wzi capsule types. This study is the first to report the efficacy of cross-reactive properties of CPS-specific Abs in both in vitro and in vivo models.

Molecular strain typing of multidrug-resistant Klebsiella pneumoniae: capsular wzi gene sequencing versus multiple locus variable number tandem repeat analysis.

This study compared genotyping of Klebsiella pneumoniae isolates by 2 molecular methods. Genotyping of 50 multidrug-resistant (MDR) and 10 non-MDR K. pneumoniae subsp. pneumoniae isolates from 2 hospitals was done using multiple locus variable number tandem repeat analysis (MLVA) and capsular typing by wzi gene sequencing. Genotyping of the isolates by the 2 methods showed 100% typeability. Agreement on clustering of the isolates by the 2 methods was 82.6%. Typing by MLVA, however, was more discriminatory (97%) than by wzi gene sequencing (92%). All the 23 K. pneumoniae subsp. pneumoniae isolates randomly selected for wzi gene sequencing showed sequence identity to previously published wzi sequences, which enabled prediction of the K-types of 16 of them. The 2 methods revealed the relatedness of (8/15) isolates from 1 of the 2 hospitals. MLVA may be considered a cheaper and more discriminatory molecular typing method suitable for genotyping of K. pneumoniae isolates in developing countries.

Molecular Epidemiology of Colonizing and Infecting Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is among the most common causes of hospital-acquired infections and has emerged as an urgent threat to public health due to carbapenem antimicrobial resistance. K. pneumoniae commonly colonizes hospitalized patients and causes extraintestinal infections such as urinary tract infection, bloodstream infection (septicemia), and pneumonia. If colonization is an intermediate step before infection, then detection and characterization of colonizing isolates could enable strategies to prevent or empirically treat K. pneumoniae infections in hospitalized patients. However, the strength of the association between colonization and infection is unclear. To test the hypothesis that hospitalized patients become infected with their colonizing strain, 1,765 patients were screened for rectal colonization with K. pneumoniae, and extraintestinal isolates from these same patients were collected over a 3-month period in a cohort study design. The overall colonization prevalence was 23.0%. After adjustment for other patient factors, colonization was significantly associated with subsequent infection: 21 of 406 (5.2%) colonized patients later had extraintestinal infection, compared to 18 of 1,359 (1.3%) noncolonized patients (adjusted odds ratio [OR], 4.01; 95% confidence interval, 2.08 to 7.73; P < 0.001). Despite a high diversity of colonizing isolates, 7/7 respiratory, 4/4 urinary, and 2/5 bloodstream isolates from colonized patients matched the patient corresponding rectal swab isolates, based on wzi capsular typing, multilocus sequence typing (MLST), and whole-genome sequence analysis. These results suggest that K. pneumoniae colonization is directly associated with progression to extraintestinal infection. IMPORTANCE K. pneumoniae commonly infects hospitalized patients, and these infections are increasingly resistant to carbapenems, the antibiotics of last resort for life-threatening bacterial infections. To prevent and treat these infections, we must better understand how K. pneumoniae causes disease and discover new ways to predict and detect infections. This study demonstrates that colonization with K. pneumoniae in the intestinal tract is strongly linked to subsequent infection. This finding helps to identify a potential time frame and possible approach for intervention: the colonizing strain from a patient could be isolated as part of a risk assessment, and antibiotic susceptibility testing could guide empirical therapy if the patient becomes acutely ill.