Environmental Assessment of SARS-CoV-2 for Internal Quality Management in a Clinical Laboratory.

BACKGROUND: As an emerging infectious disease, coronavirus disease 2019 (COVID-19) exhibits occult infection, which might cause difficulties in controlling disease spread. The possibility of aerosol transmission in a relatively closed environment contributes to the high infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in hospitals. This study presents an environmental surveillance system for SARS-CoV-2 that is suitable for a clinical laboratory and may also lead to further assessment of infection prevention programs in different departments in hospitals. METHODS: The study was performed in a SARS-CoV-2 RNA laboratory involved in the diagnosis of COVID-19 in China. Reverse transcription-polymerase chain reaction (RT-PCR) assays were used to detect viral pathogens. Standard operating procedures (SOPs) for monitoring infectious pathogens were developed in this study. RESULTS: In total, more than 180 air and surface samples were tested for SARS-CoV-2 to determine whether the virus was present at the airborne and particle level. The employed molecular method effectively identified environmental contamination. CONCLUSIONS: Our study suggests that regular environmental surveillance is critical in a clinical PCR laboratory. The presented strategy could also be used for monitoring and surveillance in negative pressure wards and clinics in hospitals to prevent hospital-acquired infections.

Characteristic and mechanism of immobilization effect of Staphylococcus aureus on human spermatozoa.

OBJECTIVE: This study was designed to investigate the impacts of Staphylococcus aureus isolated from sperm of male infertility patients, and explore the mechanism of the spermatozoa immobilization attributed to S. aureus. METHODS: S. aureus MJ015 and MJ163, the representative strains of immobilization positive and negative group respectively, were obtained from semen of infertile men. Computer-aided sperm analysis (CASA) were performed to measure sperm motility. Transmission electron microscopy (TEM) was utilized to assess morphological alterations of spermatozoa. Two-dimensional gel electrophoresis (2-DE) and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) were undertaken to analyse the difference between the secretory proteins of MJ015 and MJ163. RESULTS: A highly significant decline in motility of spermatozoa after incubating with cultured supernatant of MJ015 by sperm motility measurements, which was not observed when co-cultured with the supernatant of MJ163. TEM illustrated that the culture supernatant of MJ015 contributed to apparently ultrastructural impairment and inhibitory impacts on sperm motility. Various proteins expressed by two samples were identified. Data processing and database search preliminarily establish a link between four differential proteins and spermatozoal immobilization ability. CONCLUSIONS: Our data manifested that the clinical isolates of S. aureus have a key role on the motility and morphology of sperm. A better correlation between four identified differentially expressed proteins and the marked decline of the motility of spermatozoa was established.

A high incidence and coexistence of multiresistance genes cfr and optrA among linezolid-resistant enterococci isolated from a teaching hospital in Wenzhou, China.

Linezolid is considered as a last-resort antimicrobial agent, the resistance of which is of great concern. The aim of this study was to investigate the mechanisms and transferability of linezolid resistance and molecular epidemiology of linezolid-resistant enterococcal isolates in Wenzhou, China. A collection of 1623 enterococcal strains, including 789 Enterococcus faecalis and 834 Enterococcus faecium, were isolated from our hospital during 2011-2016. Antimicrobial susceptibility testing and clinical data analysis were performed. Molecular mechanisms of linezolid resistance, including the existence of resistance genes cfr and optrA, as well as the mutations in 23S rRNA and ribosomal proteins L3, L4, and L22, were investigated by PCR and sequencing. Conjugation experiments were conducted, and epidemiological characteristics were analyzed by PFGE and MLST. In our study, 31 (3.93%) E. faecalis and 2 (0.24%) E. faecium exhibited resistance to linezolid. Risk factors correlated with linezolid-resistant enterococcal infections included gastrointestinal surgery hospitalization, urogenital disorders, tumor, diabetes, and polymicrobial infections. Among these isolates, 6 (18.18%) harbored cfr, 9 (27.27%) harbored optrA, and 18 (54.55%) co-harbored cfr and optrA. However, mutational mechanisms were not found in this study. Conjugation experiments demonstrated the transferability of cfr and optrA between Gram-positive and Gram-negative bacteria. The clone of these isolates was diverse and scattered. It is noteworthy that cfr and optrA were the main mechanisms of linezolid resistance in this study, posing a potential risk of spread of linezolid resistance. Strikingly, it reported firstly that the two transferable resistance genes cfr and optrA coexisted in the same E. faecalis isolates.

An Uncommon ST1224 NDM-1-Producing Klebsiella pneumoniae Isolated from the Bloodstream of a Leukemia Patient in China.

AIMS: This study aimed to analyze the clinical data and characteristics of an NDM-1 (New Delhi metallo-ß-lactamase-1)-producing Klebsiella pneumoniae isolated from the bloodstream of a leukemia patient. MATERIALS AND METHODS: A retrospective analysis was used for the clinical data of the patient. The modified Hodge test (MHT) and ethylenediaminetetraacetic acid (EDTA)-disk synergy test were used for detecting metallo-ß-lactamase. Antibiotic resistance was determined using the agar dilution method. PCR was used to identify resistance genes. S1-PFGE (S1 nuclease/pulsed-field gel electrophoresis) and Southern blot hybridization were performed to determine the location of blaNDM-1. A conjugation experiment was used to confirm the transferable characteristics of the resistant genes. Multilocus sequence typing (MLST) was also performed. RESULTS: The patient developed bloodstream infections caused by this NDM-1-producing strain and died due to worsening of the condition. The strain was highly resistant to ß-lactam antibiotics and coharbored blaNDM-1, qnrB, and blaCTX-M-9 genes. Southern blot confirmed that blaNDM-1 was located on a plasmid of approximately 55 kb and could be transferred to Escherichia coli J53. MLST analysis showed that this strain belonged to an uncommon sequence type ST1224. CONCLUSION: The coexistence of various resistant genes is the mechanism for resistance to most antibiotics. Additionally, infections caused by multi-drug resistant bacteria increase the mortality of patients with immunodeficiency, which alerts clinicians to establish a rational and effective combination drug therapy.

Antifungal Susceptibility and Biological Characteristics of Fonsecaea monophora Causing Cerebral Phaeohyphomycosis in Jinhua, China.

Background: The management of cerebral abscesses caused by dark-pigmented Fonsecaea monophora in healthy individuals continues to be challenging due to no consensus on the therapeutic regimen. Due to the absence of an accurate identification method, Fonsecaea species are often misidentified due to indistinct morphology features. Materials and Methods: An F. monophora strain from an immunocompetent host with cerebral abscess was collected and identified by ITS rDNA molecular sequencing. The ITS sequences of the isolate were compared with that of the other ten Chinese F. monophora isolates obtained from GenBank for difference comparison and phylogenetic analysis. Fluorescence, Gram stains, and medan lactate were used to observe the colonial morphology. Antifungal susceptibility testing was implemented to demonstrate the antibiotic susceptibility profile. Galleria mellonella larvae were used as a model to study virulence of F. monophora. Medical records and clinical data of the patient were collected and analyzed. Results: Antifungal susceptibility testing indicated that triazole antifungal drugs possess remarkable antifungal effect against F. monophora, and satisfactory antifungal effect of itraconazole was corresponding to the drug susceptibility results. Compared with the GM test, the serum G test was found to be more sensitive. The virulence and melanization in G. mellonella models for F. monophora were observed, and the death rates of infected larvae were positively related to injected concentrations of fungus. The phylogenetic tree was constructed from the ITS sequences of the clinical isolate along with ten Chinese F. monophora isolates, revealing that there is high relatedness in F. monophora strains collected from China. Conclusion: F. monophora is an important neurotropic dematiaceous fungus and increasingly causing disease in immunocompetent individuals by means of noninvasive ways. Fungal culture, stainings, and molecular methods could be utilized to identify the etiologic agent. Triazole antifungal drugs can be applied as empiric therapeutic agents for phaeohyphomycosis.

Evolutionary Analysis of Pre-S/S Mutations in HBeAg-Negative Chronic Hepatitis B With HBsAg < 100 IU/ml.

Background: Hepatitis B surface antigen (HBsAg) and viral load are important clinical indicators for antiviral therapy. Few studies have evaluated viral sequence biomarkers predicting the risk of hepatocellular carcinoma (HCC) in the stage, which show a low serological response (HBsAg < 100 IU/ml) and high viral levels (HBV DNA > 2,000 IU/ml). This study aims to determine the trend of the biological prevalence within the pre-S/S regions of special model of inactive CHB infection. Methods: We used Sanger sequencing, quantitative HBV serology (HBeAg and HBsAg), and liver function index to identify whether HBV genome sequences are associated with long-term risk of further HCC progression in special inactive CHB infection. Results: HBV sequencing analysis of 28 CHB patients with special infectious pattern showed higher genetic diversity among four opening reading frames (ORFs) (p < 0.001). However, dN/dS ratios of HBsAg and pre-C/C regions in the experimental group showed no significantly different from those in the HCC group (p = 0.06), while significantly lower in polymerase and HBxAg regions of the experimental group (p < 0.001). In addition, seven positively selected sites were identified in pre-S1, five in pre-S2, and four in S, in which five sites (128H/135Q/135R/139L/141P) were among "α" determinant. Conclusions: These mutations in the pre-S/S region might be associated with the HCC phenotype of low HBsAg expression, with the P region possibly impacting high viral loads. Increased viral diversity across the HBV genome is also associated with low levels of HBsAg. The cumulative evolutionary changes in the HBV pre-S/S regions shows that facilitate immune evasion should be monitored individually. Due to the similarity of evolutionary characteristics in HCC, low serological responses and high viremia may be associated with the risk of further disease progression.

Molecular mechanisms and epidemiology of fosfomycin resistance in enterococci isolated from patients at a teaching hospital in China, 2013-2016.

OBJECTIVES: The aim of this study was to investigate the mechanisms of fosfomycin resistance and epidemiological characteristics in fosfomycin-resistant enterococci in China. METHODS: A collection of 761 enterococcal clinical isolates from a teaching hospital in Wenzhou, China were studied. The fosfomycin susceptibility of the isolates was investigated by the agar dilution method. The isolates were also analysed for mechanisms of re fosfomycin resistance by PCR and quantitative real-time PCR. Furthermore, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to analyse the molecular epidemiological characteristics of the fosfomycin-resistant isolates. RESULTS: In this study, 0.3% (1/372) of Enterococcus faecalis and 4.9% (19/389) of Enterococcus faecium clinical isolates were found to be resistant to fosfomycin. Among the 20 fosfomycin-resistant isolates, 5 harboured the fosB gene, 10 carried multiple amino acid substitutions in MurA, and 6 showed high-level expression of the fosX gene; of note, 1 isolate simultaneously carried fosB and amino acid mutation in MurA. Furthermore, a high degree of homology in the fosfomycin-resistant enterococci was confirmed using MLST and PFGE. CONCLUSION: These finding demonstrate that the fosB gene, mutations in the fosfomycin target enzyme MurA, and a high expression level of fosX were the resistance mechanisms in these fosfomycin-resistant enterococci.

The new perspective of old antibiotic: In vitro antibacterial activity of TMP-SMZ against Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Trimethoprim-sulfamethoxazole (TMP-SMZ) is broadly administered to treat multiple infections, and the paucity of effective treatment alternatives for infections caused by Klebsiella pneumoniae has led to a renewed interest in TMP-SMZ. The aim of this study is to evaluate the antibacterial efficacy of TMP-SMZ against K. pneumoniae. METHODS: The resistance genes of K. pneumoniae clinical isolates were investigated by PCR, followed by conjugation experiments and multilocus sequence typing. RESULTS: The resistance rate of K. pneumoniae to TMP-SMZ decreased over the collection period from 26.7% (88/330) to 16.9% (56/332). The high carrying rates (173/175, 98.9%) of resistance determinants (sul genes or dfr genes) were the main mechanisms of TMP-SMZ resistance isolates, with sul1 (142/175, 81.1%) and dfrA1 (119/175, 68.0%). Only class 1 integron was detected, the prevalence of which in TMP-SMZ resistant K. pneumoniae was 63.4% (111/175). CONCLUSION: These results provided insights into the antimicrobial efficacy of TMP-SMZ against K. pneumoniae, also illustrating the wide distribution of SMZ and TMP resistance genes among resistant K. pneumoniae. Simultaneously, the present study highlights the significance of reasonable administration and effective continued monitoring.

Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae.

Introduction. Autoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation.Aim. To investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae.Methodology. A ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts.Results. AI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-ß-1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated.Conclusion. Relationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae. These inter-connected processes could be important for bacterial group behaviour and persistence.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli.

OBJECTIVE: To evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. METHODS: Sub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. RESULTS: Based on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p<0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. CONCLUSIONS: The results revealed that treatment with sub-MICs of CIP for 24h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.

Zebrafish and Galleria mellonella: Models to Identify the Subsequent Infection and Evaluate the Immunological Differences in Different Klebsiella pneumoniae Intestinal Colonization Strains.

The intestine is the main reservoir of bacterial pathogens in most organisms. Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial bacterial infections. Intestinal colonization with K. pneumoniae has been shown to be associated with an increased risk of subsequent infections. However, not all K. pneumoniae strains in the intestine cause further infection, and the distinction of the difference among strains that cause infection after colonization and the ones causing only asymptomatic colonization is unclear. In this study, we report a case of a hospitalized patient from the ICU. We screened out two intestine colonization strains (FK4111, FK4758) to analyze the subsequent infection conditions. We set up infection models of zebrafish and Galleria mellonella to establish the differences in the potential for causing subsequent infection and the immunological specificities after K. pneumoniae intestine colonization. Sudan Black B and neutral red staining results indicated that FK4758 was more responsive to neutrophil recruitment and phagocytosis of macrophages than FK4111. The results of the assessment of the organ bacterial load revealed that FK4111 and FK4758 both had the highest bacterial loads in the zebrafish intestine compared to those in other organs. However, in the zebrafish spleen, liver, and heart, the FK4758 load was significantly higher than that of FK4111. The ST37 strain FK4111, which does not produce carbapenemase, did not cause infection after colonization, whereas the ST11 strain FK4758, which produces carbapenemase, caused infection after intestinal colonization. Our finding demonstrated that not all intestinal colonization of K. pneumoniae subsequently caused infections, and the infections of K. pneumoniae after colonization are different. Therefore, the infection models we established provided possibility for the estimation of host-microbial interactions.

An Outbreak of Carbapenem-Resistant and Hypervirulent Klebsiella pneumoniae in an Intensive Care Unit of a Major Teaching Hospital in Wenzhou, China.

Carbapenem-resistant, hypervirulent Klebsiella pneumoniae (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 K. pneumoniae isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), XbaI-macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a Galleria mellonella larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% (n = 8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase bla KPC-2. Most isolates had hypervirulent genotypes being positive for 19 virulence-associated genes, including iutA (25/29, 86.2%), rmpA (27/29, 93.1%), ybtA (27/29, 93.1%), entB (29/29, 100%), fimH (29/29, 100%), and mrkD (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the Galleria mellonella model with the highest LD50 values for three isolates (<105 CFU/mL) demonstrating the degree of hypervirulence of these CR-hvKP isolates, and is discussed relative to previous outbreaks of CR-hvKP.

Phenotype and genotype alteration during adaptive evolution of Enterococcus faecalis to antimicrobials.

The current worldwide emergence of resistance to antibiotics in bacteria constitutes an important growing public health threat. The mechanisms of the emergence and dissemination of resistance remain to be elucidated. Adaptation laboratory evolution provide an approach to investigate the acquisition of de novo mutations that confers drug resistance. In our study, 3 Enterococcus faecalis clinical isolates and E. faecalis ATCC29212 were evolved resistant to 8 kinds of antimicrobials spanning five chemical classes, including ciprofloxacin, levofloxacin, gatifloxacin, penicillin, imipenem, vancomycin, chloramphenicol and gentamicin. After 10 passages for 40 days, strains exhibited high level resistance to selected drugs except for imipenem. The greatest increase was observed in those evolved to quinolones, which caused >256-fold increase in MIC compared to the wild type. Cross-resistance and collateral-sensitivity were widely found after evolution. Through genotypic analysis of quinolones resistance strains, amino acid changes were observed in the QRDR region of GyrA and ParC. Substitutions occurred at GyrA were detected as Ser84Asn/Ser84Ile/Ser84Arg/Gly106Asp. However, Substitutions in ParC were found as Ser82Ile/Glu86Lys/Glu86Gly/His105Tyr. Compared with ancestral strains, the growth rates of evolved resistant strains slowed down and the logarithmic phase was delayed >7 h. While, the biofilm formation capacity of strains was not significantly changed by evolutionary adaptation. Our data verified that long-term exposure to sub-lethal concentrations of antimicrobial agents could selectively enrich drug resistant mutant of E. faecalis, conferring to cross-resistance and collateral-sensitivity towards other antimicrobials and fitness costs. Defining these effects can provide alternative antimicrobial strategies directed to mitigate the selection of antibiotic resistant microbes.

Antimicrobial susceptibility and mechanisms of fosfomycin resistance in extended-spectrum ß-lactamase-producing Escherichia coli strains from urinary tract infections in Wenzhou, China.

Fosfomycin in combination with various antibiotics represents an excellent clinically efficacious regimen for the treatment of urinary tract infections (UTIs) caused by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli. Underlying mechanisms of fosfomycin resistance remain largely uncharacterised. To investigate the antibacterial efficacy of fosfomycin against ESBL-producing E. coli, 356 non-repetitive ESBL-producing E. coli clinical isolates were collected from urine specimens from patients with UTI in Wenzhou, China, from January 2011 to December 2015. Antimicrobial sensitivity testing indicated that 6.7% (24/356) of the ESBL-producing E. coli strains were resistant to fosfomycin. The fosA3 gene encoding a fosfomycin-modifying enzyme was detected in 20 isolates by PCR and sequencing, alone or in combination with other ESBL determinants. Conjugation experiments and Southern blotting demonstrated that 70% (14/20) of the fosA3-positive isolates possessed transferable plasmids (ca. 54.2 kb) co-harbouring the ESBL resistance gene blaCTX-M and the fosfomycin resistance gene fosA3. Among the four fosfomycin-resistant fosA3-negative E. coli isolates, three contained amino acid substitutions (Ile28Asn and Phe30Leu in MurA and Leu297Phe in GlpT). The results indicate that presence of the fosA3 gene is the primary mechanism of fosfomycin resistance in ESBL-producing E. coli isolates in Wenzhou, China. In addition, a plasmid (ca. 54.2 kb) co-harbouring fosA3 and blaCTX-M genes is horizontally transferable. Furthermore, a low degree of homology in the fosfomycin-resistant E. coli was confirmed using multilocus sequence typing (MLST), suggesting that there is no obvious phenomenon of clonal dissemination.

Extensively Drug-Resistant Klebsiella pneumoniae Causing Nosocomial Bloodstream Infections in China: Molecular Investigation of Antibiotic Resistance Determinants, Informing Therapy, and Clinical Outcomes.

The rise in diversity of antimicrobial resistance phenotypes seen in Klebsiella pneumoniae is becoming a serious antibiotic management problem. We sought to investigate the molecular characteristics and clinical implications of extensively drug-resistant (XDR) K. pneumoniae isolated from different nosocomial bloodstream infections (BSIs) patients from July 2013 to November 2015. Even in combination treatment, meropenem did not protect against mortality of BSIs patients (P = 0.015). In contrast, tigecycline in combination with other antimicrobial agents significantly protected against mortality (P = 0.016). Antimicrobial susceptibility tests, molecular detection of antibiotic resistance determinants, conjugation experiments, multilocus sequence typing (MLST), S1-PFGE, Southern blot, SDS-PAGE, immunoblot analysis, and pulsed-field gel electrophoresis (PFGE) were used to characterize these isolates. These XDR K. pneumoniae strains were resistant to conventional antimicrobials except tigecycline and polymyxin B and co-harbored diverse resistance determinants. rmtB, blaKPC-2 as well as blaCTX-M-9 were located on a transferable plasmid of ~54.2 kb and the most predominant replicon type was IncF. 23 of the 35 isolates belonging the predominant clone were found to incorporate the globally-disseminated sequence type ST11, but others including a unique, previously undiscovered lineage ST2281 (allelic profile: 4-1-1-22-7-4-35) were also found and characterized. The porins OmpK35 and OmpK36 were deficient in two carbapenemase-negative carbapenem-resistant strains, suggesting decreased drug uptake as a mechanism for carbapenem resistance. This study highlights the importance of tracking hospital acquired infections, monitoring modes of antibiotic resistance to improve health outcomes of BSIs patients and to highlight the problems of XDR K. pneumoniae dissemination in healthcare settings.

Molecular epidemiology of extensively drug-resistant Klebsiella pneumoniae outbreak in Wenzhou, Southern China.

The emergence of extensively drug-resistant (XDR) Klebsiella pneumoniae has become a major challenge worldwide. In this study, we characterized the phenotypes and genetic features of nine XDR K. pneumoniae isolates from an integrated hospital in Zhejiang province, China, from September to October 2014. These XDR K. pneumoniae possessed at least five resistance determinants which contribute to highly resistant to ß-lactam, ß-lactam/inhibitor combinations, aminoglycosides, quinolones, carbapenems, chloroamphenicol and fosfomycin. All isolates carried blaKPC-2, blaCTX-M-9, blaSHV-11 and rmtB, and several isolates also harboured blaTEM-1 and qnrS. Southern blot experiments confirmed that blaKPC-2, rmtB and blaCTX-M-9 were located on the same ~54.2 kb plasmid. Conjugative plasmids were obtained from all K. pneumoniae isolates, further proving the transferable characteristic of the resistance determinants. The OmpK36 sequences showed various deletions and insertions that indicated additional amino acid residues and a deleted phenotype of OmpK36. PFGE demonstrated that all the isolates belonged to the same genotype. Multilocus sequence typing was concordant with PFGE results and revealed that ST11 was the most predominant clone. Our study revealed a high incidence and endemic spread of XDR K. pneumoniae in the hospital. Thus, effective infection control measures should be adopted to monitor and control the spread of multidrug-resistant isolates.

Effects of sub-minimum inhibitory concentrations of ciprofloxacin on biofilm formation and virulence factors of Escherichia coli

ABSTRACT Objective: To evaluate the influence of sub-minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) on biofilm formation and virulence factors of Escherichia coli clinical isolates. Methods: Sub-MICs of CIP were determined using growth curve experiments. The biofilm-forming capacity of E. coli clinical isolates and E. coli ATCC 25922 treated or untreated with sub-MICs of CIP was assessed using a crystal violet staining assay. The biofilm structure of E. coli isolate was assessed with scanning electron microscopy (SEM). The expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were measured using quantification RT-PCR (qRT-PCR) in E. coli isolates and E. coli ATCC 25922. Results: Based on our results, the sub-MICs of CIP were 1/4 MICs. Sub-MICs of CIP significantly inhibited biofilm formation of E. coli clinical isolates and E. coli ATCC 25922 (p < 0.01). SEM analyses indicated that the biofilm structure of the E. coli changed significantly after treatment with sub-MICs of CIP. Expression levels of the virulence genes fim, usp, and iron and the biofilm formation genes of the pgaABCD locus were also suppressed. Conclusions: The results revealed that treatment with sub-MICs of CIP for 24 h inhibited biofilm formation and reduced the expression of virulence genes and biofilm formation genes in E. coli.