Ceftazidime/Avibactam Resistance in Carbapenemase-Producing Klebsiella pneumoniae.

We identified a novel ceftazidime/avibactam resistance mechanism in sequence type 11 Klebsiella pneumoniae carbapenemase 2-producing K. pneumoniae. Plasmid recombination and chromosomal integration formed a novel virulence plasmid and provided an additional promoter for blaSHV-12, leading to blaSHV-12 overexpression and ceftazidime/avibactam resistance. Genetic rearrangement contributed to convergence of hypervirulence and ceftazidime/avibactam resistance.

Dissecting the genotypic features of a fluoroquinolone-resistant Pseudomonas aeruginosa ST316 sublineage causing ear infections in Shanghai, China.

Limited information is available regarding the genomic characteristics of P. aeruginosa causing ear infections. Our aim is to characterize the genotypic features of an emerging ST316 sublineage causing aural infections in Shanghai. A total of 199 ear swab isolates were subjected to whole genome sequencing (WGS). Complete genomes for two isolates were resolved. We showed this recently emerged sublineage exhibited high-level resistance to fluoroquinolones (FQs) primarily by accumulation of known mutations in quinolone resistance determining regions (QRDRs). Loss-of-function mutations in mexR and mexCD were frequently detected. Mutations in fusA1 (P166S) and parE (S492F) were resident in this sublinage about 2 years after its emergence. Recombination events might be a key driver of genomic diversity in this sublineage. Convergent evolution events on Multidrug-resistant (MDR) determinants were also observed. We generated predictive machine models and identified biomarkers of resistance to gentamicin, fosfomycin, and cefoperazone-sulbactam in this sublineage. This sublineage tended to be less virulent by loss of a series virulence genes represented by ppkA, rhlI, and iron uptake- and antimicrobial activity-related genes. Specific mutations were detected in pilU and lpxB genes that related to surface structures. Moreover, this sublineage differed from non-ST316 isolates in several ways, including virulence genes related to cell surface structure. Our analysis suggested acquisition of a roughly 390 kbp MDR plasmid carrying qnrVC1 might play an important role in the success of this sublinage. Clonal expansion of this sublineage exhibiting enhanced adaptation to cause ear infections is concerning, which requires urgent control measures to be implemented.

Antibiotic Exposure during the Preceding Six Months Is Related to Intestinal ESBL-Producing Enterobacteriaceae Carriage in the Elderly.

Intestinal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE carriage) poses a health risk to the elderly. It was aimed to study the prevalence and the risk factors of intestinal ESBL-PE carriage in the elderly. An observational study of a 921-elderly cohort was examined at health checkup for intestinal ESBL-PE carriage at a tertiary medical center in Shanghai. The prevalence and risk factors of intestinal ESBL-PE carriage, especially antimicrobial use in the preceding 9 months, were studied. The prevalence of intestinal ESBL-PE carriage was 53.3% (491/921) in community-dwelling elderly people. A total of 542 ESBL-producing isolates, including E. coli (n = 484) and K. pneumoniae (n = 58), were obtained. On genotyping, the CTX-M-9 ESBL was the most prevalent for 66.0% (358/542) of all isolates. Multivariate analysis showed that antibiotic exposure, age (61-70 years), and nursing home residence were independent risk factors of the ESBL-PE carriage. The analysis on the monthly use of antimicrobials showed that antibiotic exposure during the 6 months prior to sample collection contributed to the high prevalence of ESBL-PE carriage. A single exposure to an antimicrobial increased the risk of the carriage significantly, and the risk increased with the frequency of antimicrobial exposure (RR, 1.825 to 5.255). Prior use of second or third generation cephalosporins, fluoroquinolones, and macrolides increased the risk of the carriage. The results of this study indicate the importance of using antimicrobials judiciously in clinical settings to reduce antimicrobial resistance. Further studies with multiple center surveillance and with comparison of ESBL-PE carriage in the elderly and in the general population simultaneously are needed.

The Vancomycin Resistance-Associated Regulatory System VraSR Modulates Biofilm Formation of Staphylococcus epidermidis in an ica-Dependent Manner.

The two-component system VraSR responds to the cell wall-active antibiotic stress in Staphylococcus epidermidis. To study its regulatory function in biofilm formation, a vraSR deletion mutant (ΔvraSR) was constructed using S. epidermidis strain 1457 (SE1457) as the parent strain. Compared to SE1457, the ΔvraSR mutant showed impaired biofilm formation both in vitro and in vivo with a higher ratio of dead cells within the biofilm. Consistently, the ΔvraSR mutant produced much less polysaccharide intercellular adhesin (PIA). The ΔvraSR mutant also showed increased susceptibility to the cell wall inhibitor and SDS, and its cell wall observed under a transmission electron microscope (TEM) appeared to be thinner and interrupted, which is in accordance with higher susceptibility to the stress. Complementation of vraSR in the ΔvraSR mutant restored the biofilm formation and the cell wall thickness to wild-type levels. Transcriptome sequencing (RNA-Seq) showed that the vraSR deletion affected the transcription levels of 73 genes, including genes involved in biofilm formation, bacterial programmed cell death (CidA-LrgAB system), glycolysis/gluconeogenesis, the pentose phosphate pathway (PPP), and the tricarboxylic acid (TCA) cycle, etc. The results of RNA-Seq were confirmed by quantitative real-time reverse transcription-PCR (qRT-PCR). In the ΔvraSR mutant, the expression of icaA and lrgAB was downregulated and the expression of icaR and cidA was upregulated, in comparison to that of SE1457. The transcriptional levels of antibiotic-resistant genes (pbp2, serp1412, murAA, etc.) had no significant changes. An electrophoretic mobility shift assay further revealed that phosphorylated VraR bound to the promoter regions of the ica operon, as well as its own promoter region. This study demonstrates that in S. epidermidis, VraSR is an autoregulator and directly regulates biofilm formation in an ica-dependent manner. Upon cell wall stress, it indirectly regulates cell death and drug resistance in association with alterations to multiple metabolism pathways. IMPORTANCE S. epidermidis is a leading cause of hospital-acquired catheter-related infections, and its pathogenicity depends mostly on its ability to form biofilms on implants. The biofilm formation is a complex procedure that involves multiple regulating factors. Here, we show that a vancomycin resistance-associated two-component regulatory system, VraSR, plays an important role in modulating S. epidermidis biofilm formation and tolerance to stress. We demonstrate that S. epidermidis VraSR is an autoregulated system that selectively responds to stress targeting cell wall synthesis. Besides, phosphorylated VraR can bind to the promoter region of the ica operon and directly regulates polysaccharide intercellular adhesin production and biofilm formation in S. epidermidis. Furthermore, VraSR may indirectly modulate bacterial cell death and extracellular DNA (eDNA) release in biofilms through the CidA-LrgAB system. This work provides a new molecular insight into the mechanisms of VraSR-mediated modulation of the biofilm formation and cell death of S. epidermidis.

Molecular Characteristics, Antimicrobial Resistance, and Biofilm Formation of Pseudomonas aeruginosa Isolated from Patients with Aural Infections in Shanghai, China.

PURPOSE: To investigate molecular characteristics, antimicrobial resistance, and biofilm formation ability of Pseudomonas aeruginosa strains isolated from patients with aural infections. METHODS: Isolates (n = 199) were collected from ear discharges of patients with aural infections from January 2019 to December 2020. Antimicrobial susceptibility testing was performed according to the Clinical and Laboratory Standards Institute guidelines. All isolates were subjected to multilocus sequence typing (MLST) with amplification and sequencing of seven housekeeping genes. Biofilm formation and eradication were quantitatively assessed in microtiter plates. Genes associated with biofilm formation and the quinolone-resistance-determining region (QRDR) of genes gyrA and parC were investigated using polymerase chain reaction amplification and sequencing. RESULTS: Of the 199 P. aeruginosa strains isolated, 109 (54.77%) were from females and 90 (45.23%) were from males. The isolates exhibited very low rates of resistance to most antibiotics tested, including piperacillin (1.51%), ceftazidime (0.50%), and imipenem (3.52%); however, the quinolones ciprofloxacin (80.40%) and levofloxacin (82.91%) were notable exceptions. The QRDR sequence results of the quinolone-resistant P. aeruginosa isolates showed Thr83Ile (n = 155) was the most common amino acid mutation in gyrA (n = 165), while Ser87Leu (n = 157) was widely detected in parC (n = 165). MLST analysis identified 34 sequence types (STs) with most isolates belonging to ST316 (73.87%). Almost all of the P. aeruginosa isolates (96.98%) produced biofilms and biofilm-forming genes algD (98.49%), pslD (96.98%), and pelF (96.48%) were highly prevalent. CONCLUSION: The P. aeruginosa strains isolated from aural discharges in this study exhibited very low rates of resistance to most antibiotics tested, except for the resistance rates to quinolones, which were relatively high. The isolates also exhibited a strong biofilm formation ability and low susceptibility to eradication, indicating that more effective drugs and treatment methods are needed to combat these infections.

Microbial Spectrum and Resistance Patterns in Ocular Infections: A 15-Year Review in East China.

PURPOSE: To report antibiotic resistance rates and trends of common ocular isolates collected over a 15-year period. METHODS: We collected 3533 isolates from July 1, 2005 to July 31, 2020. Antibiotic sensitivity was determined according to the guidelines of the Clinical and Laboratory Standards Institute. Chi-squared (χ 2) test was used to analyze changes in antibiotic susceptibility over 15 years. RESULTS: Among the 3533 isolates, the predominant pathogens were the staphylococcal species. Methicillin resistance was observed in 381 Staphylococcus aureus (S. aureus) isolates (46.4%) and 1888 coagulase-negative staphylococci (CoNS) isolates (61.1%), and methicillin-resistant (MR) isolates had a high probability of concurrent resistance to fluoroquinolones and aminoglycosides. The mean percentage of resistance in staphylococcal isolates did not reach statistical significance across patient age groups (P = 0.87). Methicillin resistance did not increase in the CoNS (P = 0.546) isolates, and resistance to methicillin slightly decreased among S. aureus (P = 0.04) isolates over 15 years. Additional exploratory analysis revealed a small decrease in resistance to tobramycin (P = 0.01) and chloramphenicol (P < 0.001) among the CoNS isolates. All staphylococcal isolates were susceptible to vancomycin. CONCLUSION: Staphylococci were the most common microorganisms responsible for causing ocular infections. Antibiotic resistance was high among staphylococci, with nearly half of these isolates were resistant to methicillin and these had a high probability of concurrent resistance among MR staphylococci to other antibiotics. Overall, ocular resistance did not significantly change during the 15-year study period. We conclude that continued surveillance of antibiotic resistance provides critical data to guide antibiotic selection.

The Colonization of Carbapenem-Resistant Klebsiella pneumoniae: Epidemiology, Resistance Mechanisms, and Risk Factors in Patients Admitted to Intensive Care Units in China.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a threat to public health, most notably as a superbug causing nosocomial infections. Patients in the intensive care unit (ICU) are at increased risk of hospital-acquired K pneumoniae infection, especially CRKP. This study was conducted to investigate the frequency of gastrointestinal and nasopharyngeal K pneumoniae colonization and its contribution to infections in ICU patients. METHODS: A 3-month prospective cohort study was performed in which 243 ICU patients were screened for intestinal and nasopharyngeal carriage of K pneumoniae at admission and once per week thereafter. The colonization and clinical infection isolates were analyzed by antimicrobial susceptibility testing to identify CRKP and were characterized by multilocus sequence typing (MLST) and whole-genome sequencing combined with epidemiological data to investigate the resistance mechanisms and assess the possible transmitted infection. RESULTS: Twenty-eight percent (68 of 243) of patients tested positive for carriage of K pneumoniae immediately upon admission to ICU, 54% (37 of 68) of which were nonduplicate CRKP isolates. Patients with carbapenem-susceptible K pneumoniae (CSKP) colonization at admission were more likely to acquire CRKP colonization during the ICU stay compared with patients without K pneumoniae colonization at admission. The incidence of subsequent CRKP infection in the baseline CSKP (32.3%, 10 of 31) and CRKP (45.9%, 17 of 37) carrier group was significantly higher than that of the baseline non-KP carrier group (8.6%, 15 of 175). The risk factors associated with acquired CRKP colonization during the ICU stay among negative CRKP colonization at admission included previous exposure to carbapenem, tigecycline or ß-lactam/ß-lactamases inhibitor, and invasive processes or surgical operations. Sixty-four percent (27 of 42) of patients with K pneumoniae infection were colonized by clonally related K pneumoniae strains according to enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction analysis. ST11 (72%, 53 of 74) was the most predominant MLST type of clonally related CRKP isolate colonizing these patients, followed by ST15 (26%, 19 of 74). CONCLUSIONS: The colonization of K pneumoniae may increase the incidence of corresponding K pneumoniae infection in critically ill patients in the ICU. High prevalence of ST11 CRKP (due to blaKPC-2) carriage and infection in ICU was observed.

The Predominance of Strain Replacement Among Enterobacteriaceae Pairs With Emerging Carbapenem Resistance During Hospitalization.

Isolates of Enterobacteriaceae collected from the same patient can lose carbapenem susceptibility during antimicrobial therapy, but little attention has been given to how this conversion takes place. In the current study, we retrospectively analyzed microbiological and clinical data from patients with enterobacterial infections at a tertiary hospital in Shanghai, China. After screening 4795 patients and 7120 Enterobacteriaceae isolates over the 3-year study period, we found the change from carbapenem susceptible to carbapenem resistant in 41 pairs of isolates, of which 35 pairs (85.4%) were K. pneumoniae and 25 (61.0%) were from the same anatomic sites. Thirty-six isolate pairs showed different pulsed-field gel electrophoresis patterns between the carbapenem-susceptible and the corresponding resistant strain, and 5 pairs displayed identical pulsed-field gel electrophoresis patterns. Thirty-three (91.7%) of the 36 pairs of Enterobacteriaceae isolates were carbapenem-resistant K. pneumoniae with blaKPC-2, and 28 pairs (90.3%) of K. pneumoniae isolates had different sequence types (STs), with ST11 the most common ST found in carbapenem-resistant K. pneumoniae isolates. Forty of the 41 patients had received antimicrobial therapy such as carbapenems, cephalosporins, and fluoroquinolones, before the isolation of carbapenem-resistant Enterobacteriaceae. These results demonstrated that strain replacement is the main cause of emerging carbapenem resistance in Enterobacteriaceae during hospitalization. The loss of carbapenem susceptibility was not mainly due to in vivo development of carbapenem resistance.

Isobaric tags for relative and absolute quantitation proteomics analysis of gene regulation by SprC in Staphylococcus aureus.

AIM: To explore the complete gene networks regulated by small RNA SprC and its targets in Staphylococcus aureus. MATERIALS & METHODS: The isobaric tags for relative and absolute quantitation and bioinformatic methods were utilized to identify and analyze the target proteins affected by SprC in S. aureus N315. RESULTS: Proteomic analysis showed that the expression of 44 proteins was modulated by SprC. Further, bioinformatic analysis displayed that these affected proteins mainly associated with metabolic and cellular process, biological regulation and catalytic activity. CONCLUSION: Our data provide a rich resource of SprC targets in S. aureus, although the mechanism of regulation by SprC is yet to be elucidated.

In vivo development of tigecycline resistance in Klebsiella pneumoniae owing to deletion of the ramR ribosomal binding site.

Tigecycline resistance is emerging among Klebsiella pneumoniae, but knowledge regarding in vivo development of tigecycline resistance is limited. Here we report a new mechanism of tigecycline resistance in K. pneumoniae that evolved during tigecycline therapy. Klebsiella pneumoniae isolates were consecutively obtained from urine samples of a patient with scrotal abscess and urinary tract infection before and during tigecycline treatment. Two tigecycline-resistant K. pneumoniae strains (KP-3R and KP-4R; MIC = 8 µg/mL) were isolated after 41 days and 47 days of tigecycline therapy. These isolates had the same sequence type (ST11) and PFGE patterns as tigecycline-susceptible strains (KP-1S and KP-2S; MIC = 2 µg/mL) initially isolated from the patient. Compared with KP-1S and KP-2S, KP-3R and KP-4R exhibited higher expression of efflux pump AcrAB. Sequence comparison of the repressor gene ramR did not find any mutation within the open-reading frame that exist frequently in tigecycline-resistant K. pneumoniae. Instead, a 12-bp deletion of ramR upstream region including the ribosomal binding site (RBS) TGAGG was observed in KP-3R and KP-4R. qRT-PCR and immunoblotting analyses showed that KP-3R and KP-4R did not have impaired ramR transcription but had abolished RamR protein production. Furthermore, xylE reporter assay demonstrated that KP-3R and KP-4R had a defect in RamR translation caused by the 12-bp deletion. Complementing KP-3R and KP-4R with functional ramR suppressed expression of acrAB and consequently restored tigecycline susceptibility. This is the first report identifying deletion of the ramR RBS as a mechanism of in vivo tigecycline resistance in K. pneumoniae developing during tigecycline therapy.

High ceftazidime hydrolysis activity and porin OmpK35 deficiency contribute to the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae.

Objectives: To investigate mechanisms for the decreased susceptibility to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae (KPC-KP). Methods: A total of 24 isolates, 8 each with ceftazidime/avibactam MICs of 4-8, 1-2 and ≤0.5 mg/L, were randomly selected from 214 clinical isolates of KPC-KP, and the ß-lactamase hydrolysis activity and porin expression profiles were determined. Plasmid profile and relative expression and copy number of the bla KPC gene were also analysed. Results: Ceftazidime/avibactam MIC 50 and MIC 90 were 2 and 4 mg/L, respectively, for the 214 KPC-KP isolates. The hydrolysis activities of nitrocefin and ceftazidime in both of the ceftazidime/avibactam MIC 4-8 and 1-2 mg/L groups were significantly higher than those of the MIC ≤0.5 mg/L group, while the hydrolysis activities were 4-4.6-fold higher in the MIC 4-8 mg/L group than in the other two groups when 4 mg/L avibactam was added. The relative expression and copy number of the bla KPC gene in the MIC 4-8 mg/L group were 4.2-4.8-fold higher than in the other two groups. Meanwhile, SDS-PAGE showed that all isolates in the two groups with MIC ≥1 mg/L lacked OmpK35, which had either an early frameshift with a premature stop codon ( n = 15, ST11) or overexpression of the negative regulation genes, micF and ompR ( n = 1, ST15), whereas OmpK35 and OmpK36 could both be observed in all isolates with MIC ≤0.5 mg/L. Conclusions: Decreased ceftazidime/avibactam susceptibility in KPC-KP clinical isolates is caused by high ceftazidime hydrolysis activity and OmpK35 porin deficiency and the majority of isolates belong to ST11.

CTX-M-190, a Novel ß-Lactamase Resistant to Tazobactam and Sulbactam, Identified in an Escherichia coli Clinical Isolate.

A novel ß-lactamase, CTX-M-190, derived from CTX-M-55 by a single substitution of Ser for Thr at position 133 (Ser133Thr), was identified in a natural Escherichia coli clinical isolate. CTX-M-190 exhibited potent hydrolytic activity against cefotaxime, with a kcat/Km ratio of 14.5 µM-1 s-1, and was highly resistant to inhibition by the ß-lactamase inhibitors tazobactam and sulbactam, whose 50% inhibitory concentrations were 77- and 55-fold higher, respectively, for CTX-M-190 than for CTX-M-55. blaCTX-M-190 was located within the genetic platform ISEcp1-blaCTX-M-orf477, which was harbored by a 70-kb IncI1 plasmid.

Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes.

Hypervirulent variants of Klebsiella pneumoniae (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80-100% among LA-Kp isolates while 2-11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.

In vivo Acquisition of Carbapenemase Gene blaKPC-2 in Multiple Species of Enterobacteriaceae through Horizontal Transfer of Insertion Sequence or Plasmid.

Objectives: Current worldwide spread of carbapenem resistance in Enterobacteriaceae constitutes a critical public health threat. This study aims to investigate how carbapenem resistance is acquired in Enterobacteriaceae in patients during antimicrobial therapy. Methods: Clinical strains from the same anatomical site of the same patients that converted from carbapenem-susceptible to resistant during antimicrobial therapy and showed identical or similar PFGE patterns were identified. The similarly sized plasmids carried by the susceptible and resistant strains, the latter containing the carbapenemase genes, were sequenced and analyzed. Results: Paired strains were identified from four patients: three had neurosurgical conditions while the other had acute exacerbation of COPD. Two pairs of Klebsiella pneumoniae (KP1-S/R and KP2-S/R, S and R indicating susceptible and resistant strains, respectively), one pair of Morganella morganii (MM-S/R) and one pair of Enterobacter aerogenes (EA-S/R) were collected. All four carbapenem-resistant strains carried plasmids harboring blaKPC-2. Compared with the similarly sized plasmids in KP1-S and KP2-S, an insertion sequence that includes ISKpn6-like, blaKPC-2 and ISKpn8 was noted in pKP1-R and pKP2-R. Strains MM-R and EA-R had blaKPC-2-carrying plasmids not resembling plasmids in strains MM-S and EA-S suggesting their new acquisition while on therapy. Conclusions: Enterobacteriaceae can acquire carbapenem resistance during antimicrobial therapy through horizontal transfer of an insertion sequence or plasmid.

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Characterization of a Novel IncHI2 Plasmid Carrying Tandem Copies of blaCTX-M-2 in a fosA6-Harboring Escherichia coli Sequence Type 410 Strain.

The extended-spectrum ß-lactamase gene blaCTX-M-2 is mainly associated with ISCR1 embedded in complex sul1-type integrons, but information on the genetic context of plasmids harboring the ISCR1-blaCTX-M-2 module remains limited. In this study, a blaCTX-M-2-harboring plasmid (pYD786-1) belonging to the sequence type 2 (ST2)-IncHI2 plasmid type and isolated from an Escherichia coli ST410 clinical strain was sequenced and analyzed. pYD786-1 belongs to the APEC-O1-R-type IncHI2 plasmids, which are widely distributed in human, poultry, and livestock strains. It contains a multidrug resistance mosaic region (MRR) consisting of a Tn21::In2 transposon backbone augmented by acquisition of duplicate ISCR1-blaCTX-M-2 modules. Tn2411, a Tn21::In2 precursor, likely played a role in the generation of the MRR in pN13-01290_23, the putative progenitor plasmid of pYD786-1, found in a foodborne Salmonella strain. Tn21/Tn2411::In::ISCR1-blaCTX-M-2 derivatives, including pYD786-1, have been identified in strains from Europe, South America, and the United States, suggesting potential global dissemination of the blaCTX-M-2 modules mediated by this vehicle.

Four carbapenem-resistant gram-negative species carrying distinct carbapenemases in a single patient.

Carbapenem-resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, and Acinetobacter baumannii were isolated from a single patient, each producing different carbapenemases (NDM-1, KPC-2, IMP, and OXA-23, respectively). The NDM-1-producing E. coli strain was preceded by a clonally related carbapenem-susceptible strain a month earlier, suggesting in vivo acquisition of blaNDM-1.

Klebsiella pneumoniae: Development of Carbapenem Resistance due to Acquisition of bla NDM-1 During Antimicrobial Therapy in Twin Infants with Pneumonia.

OBJECTIVES: To identify the mechanism of in vivo development of carbapenem resistance in Klebsiella pneumoniae. METHODS: Seven sequential isolates of K. pneumoniae were obtained from twin infants with pneumonia. Antimicrobial susceptibility testing was performed by agar dilution method. Carbapenemases including KPC and MßL were initially screened using phenotypic methods, and carbapenemase-encoding genes were identified by polymerase chain reaction and amplicon sequencing. Plasmids of all clinical isolates and the conjugants of resistant isolates were estimated by S1 pulsed-field gel electrophoresis (PFGE). Molecular typing were conducted by PFGE of XbaI-digested genomic DNA and multilocus sequence typing. RESULTS: For old brother, the first and third isolates were susceptible to meropenem, whereas the second and fourth isolates were resistant (MICs 16 mg/L). The first and second isolates from the young brother were susceptible to meropenem whereas the third isolate was resistant. All the resistant isolates produced NDM-1 metallo-ß-lactamase. PFGE of XbaI-digested DNA revealed almost identical patterns with similarity indices of above 92% for all the seven isolates. All the isolates had the same sequence type named sequence type 37 (ST37). CONCLUSION: To our knowledge, this is the first documented case of development of carbapenem resistance in vivo mediated by NDM-1 metallo-ß-lactamase in K. pneumoniae during treatment of pneumonia with meropenem.

Prevalence of fusB in Staphylococcus aureus clinical isolates.

Fusidic acid (FA) resistance in Staphylococcus aureus markedly varied among different regions. Few data for FA resistance are available in China. In this study, FA susceptibility testing was performed, and the prevalence of fusB and fusC in 116 clinical isolates of S. aureus was investigated by PCR. Mutations in fusA were also determined by sequencing of PCR products. Molecular typing of fusB-positive strains was based on multilocus sequence typing (MLST), spa typing and pulsed-field gel electrophoresis (PFGE). A DNA fragment flanking fusB was sequenced. Transformation experiments were carried out in fusB-positive S. aureus. Of 116 S. aureus including 19 meticillin-resistant S. aureus (MRSA) and 97 meticillin-susceptible S. aureus (MSSA), four (3.5 %) were resistant to FA with MICs of 6-12 µg ml(-1), including one MRSA from blood and three MSSA from wound exudates. All four FA-resistant isolates were found to be fusB gene positive. Three FA-resistant MSSA strains had the same MLST profile of ST630 and spa type of t377, whilst the MRSA strain belonged to ST630-t4549. Only one PFGE pattern was recognized for these four strains. No fusC and fusA mutations were detected in any of the isolates. FA resistance in fusB-positive clinical isolates could be transferred to S. aureus RN4220. The fusB gene was located in a transposon-like element, which had 99 % identity with that found in pUB101. In conclusion, the FA resistance rate is low in S. aureus, and the fusB gene is responsible for the resistance.

Outbreak of pulmonary infection caused by Klebsiella pneumoniae isolates harbouring blaIMP-4 and blaDHA-1 in a neonatal intensive care unit in China.

Outbreaks caused by Klebsiella pneumoniae producing carbapenemases and other ß-lactamases have been reported. Four neonates admitted to a neonatal intensive care unit (NICU) in a Chinese hospital developed respiratory infection while receiving intensive care. In all four cases, multidrug-resistant K. pneumoniae was isolated from multiple respiratory specimens, leading to additional characterization of these organisms and investigation of the local environment in the NICU. Multiple ß-lactamase genes, including bla(TEM-1), bla(IMP-4), bla(DHA-1) and bla(CTX-M-14), as well as the quinolone resistance gene qnrB4, were harboured by transferable plasmids from all four clinical isolates. Furthermore, PFGE confirmed that three of the four clinical isolates from the patients and three K. pneumoniae isolates collected from the hands of health-care workers and an incubator in the NICU belonged to the same PFGE cluster, indicating that an outbreak due to multidrug-resistant K. pneumoniae carrying bla(IMP-4) and bla(DHA-1) occurred in this NICU. As far as is known, this is the first report of the co-existence of bla(IMP-4) and bla(DHA-1) in the same K. pneumoniae isolate. These data suggest that additional precautions are needed to prevent outbreaks of infection caused by multidrug-resistant K. pneumoniae resulting from environmental exposure in NICUs.