OXA-51-like β-lactamases and their association with particular epidemic lineages of Acinetobacter baumannii

Sixty diverse clinical Acinetobacter baumannii isolates of worldwide origin were assigned to sequence groups, based on a multiplex PCR for the ompA , csuE and bla _OXA-51-like genes. The majority (77%) of isolates belonged to sequence groups 1 and 2 (SG1 and SG2), with sequence group 3 (SG3) and non-grouped isolates accounting for the remainder. The isolates were not closely related according to pulsed-field gel electrophoresis (PFGE), and the majority were sensitive to imipenem and meropenem. The construction of a linkage map of OXA-51-like β-lactamase sequence relationships revealed two closely related clusters of enzymes, one focused around OXA-66 and the other around OXA-69. Isolates belonging to SG1 encoded an enzyme from the OXA-66 cluster, while those belonging to SG2 encoded an enzyme from the OXA-69 cluster. All SG3 isolates encoded OXA-71, which does not form part of a close enzyme grouping. Major multinational lineages accounted for a significant proportion of A. baumannii clinical isolates, and the evolution of the OXA-51-like enzymes appears to be an ongoing process.     

Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system

Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-β-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.     

First identification of an Escherichia coli clinical isolate producing both metallo-β-lactamase VIM-2 and extended-spectrum β-lactamase IBC-1

An Escherichia coli strain with decreased susceptibility to carbapenems was isolated from a hospitalised patient in Athens, Greece. The strain was resistant to all beta-lactams, including aztreonam, whereas the MIC of imipenem and meropenem was 0.5 mg/L. A positive EDTA-disk synergy test suggested the production of a metallo-beta-lactamase. PCR experiments revealed the presence of the bla(VIM-2), bla(IBC-1), and bla(TEM-1) genes. Resistance to beta-lactams was not transferable by conjugation. This is the first report of a clinical isolate of E. coli producing VIM-2, and the first report of the coexistence of bla(VIM-2) and bla(IBC-1) in a single clinical isolate.     

Amoxicillin resistance with β-lactamase production in Helicobacter pylori

Background Amoxicillin‐resistant Helicobacter pylori with minimal inhibitory concentration (MIC) ≥ 256 mg L^?1 was isolated from a gastritis patient. The aims were to investigate the mechanism of high‐level amoxicillin resistance in H. pylori. Materials and methods The β‐lactamase production was determined by means of nitrocefin sticks and the presence of gene encoding the β‐lactam antibiotic resistance enzyme TEM β‐lactamase was analysed by polymerase chain reaction (PCR), sequencing and dot‐blot hybridization. Sequencing analysis of pbp1A gene was performed and amoxicillin‐susceptible isolate was transformed with pbp1A PCR products from the resistant isolate. The expression of hefC efflux system was analysed using real‐time quantitative PCR. Results Activity of β‐lactamase was detected. Sequence analysis showed that the PCR product derived from H. pylori 3778 was identical to the bla_TEM‐1 (GenBank accession EU726527 ). Dot‐blot hybridization confirmed the presence of β‐lactamase gene bla_TEM‐1. By transformation of PCR product of mutated pbp1A gene from H. pylori 3778 into amoxicillin‐susceptible strain showed that substitutions in Thr₅₅₆→Ser, Lys₆₄₈→Gln, Arg₆₄₉→Lys and Arg₆₅₆→Pro contribute to low‐level amoxicillin resistance. The MIC of amoxicillin for the transformants was 0·75 mg L^?1. Over‐expression of hefC was not found. Conclusions High‐level amoxicillin resistance is associated with β‐lactamase production in H. pylori. Low‐level amoxicillin resistance is linked to a point mutation on pbp1A. Because H. pylori can exchange DNA through natural transformation, spreading of bla_TEM‐1 amoxicillin resistance gene among H. pylori is a potential threat when treating H. pylori infection.     

316株儿童分离流感嗜血杆菌的耐药性和β-内酰胺酶基因分型

摘要：目的 探讨儿童分离流感嗜血杆菌对抗菌药物的耐药情况和β-内酰胺酶基因分型。方法 收集2016年上海市儿童医院临床分离的流感嗜血杆菌316株；纸片扩散法检测细菌对抗菌药物的耐药性，头孢硝噻吩纸片检测细菌β-内酰胺酶的表型，PCR方法对细菌β-内酰胺酶基因进行检测分型。结果 316株流感嗜血杆菌对甲氧苄啶/磺胺甲噁唑耐药率67.7%，氨苄西林52.2%，阿奇霉素28.5%，头孢呋辛26.9%，氨苄西林/舒巴坦10.8%。对头孢噻肟、阿莫西林/克拉维酸敏感率均>90%。41.1%（130/316）检测到β-内酰胺酶基因，与表型一致，基因分型均为TEM-1型，未检测到ROB-1型。结论 三代头孢和加酶抑制剂抗菌药物（头孢噻肟和氨苄西林/舒巴坦、阿莫西林/克拉维酸）对儿童感染流感嗜血杆菌有较好抗菌活性，可供临床用药参考。流感嗜血杆菌β-内酰胺酶基因分型均为TEM-1型。     

Direct screening of the IMP-1 metallo-β-lactamase gene (blaIMP) from urine samples by polymerase chain reaction

BACKGROUND: As Gram-negative bacterial isolates producing plasmid-mediated IMP-1 metallo-beta-lactamase usually demonstrate resistance to various broad-spectrum beta-lactams, including cephamycins and carbapenems, transmission and proliferation of these microorganisms in clinical settings could become a clinical threat in the near future. According to previous studies by the same authors, IMP-1-producing strains are usually isolated from urine samples. Therefore, in this study, a polymerase chain reaction (PCR) was applied for direct screening of the IMP-1 metallo-beta-lactamase gene in urine samples. METHOD: Urine samples were collected from 273 inpatients to whom various broad-spectrum beta-lactams, including carbapenems, had been administered in 57 hospitals in 1997. DNA templates for PCR analyses were prepared directly from 19 urine samples from which Serratia marcescens strains demonstrating high-level resistance (minimal inhibitory concentration > 128 microg/mL) to both ceftazidime and cefoperazone-sulbactam were later isolated. RESULTS: The IMP-1 metallo-beta-lactamase gene (blaIMP)-specific 578 bp fragments were able to be amplified successfully in eight of the 19 samples. In the seven strains isolated from the eight samples, the presence of blaIMP was also detected by a DNA hybridization analysis. The lower limit of the PCR method was determined as 1 x 10(2) CFU of blaIMP-bearing bacterial cells per 1 mL of urine sample. No false positive result was found. CONCLUSION: The PCR-aided direct screening of blaIMP is applicable to early recognition of IMP-1-producing bacteria in urine samples. This method would help to prevent nosocomial and interhospital transmission of this kind of hazardous bacteria, as well as the advancement of rigorous infection control.     

Genetic association of blaSHV-5 with transposable elements IS26 and IS5 in Klebsiella pneumoniae from Taiwan

A cloned 5,248-bp EcoRI fragment from the Klebsiella pneumoniae transferable plasmid pKP53 (> 70 kb) containing bla(SHV-5) was sequenced. Insertion sequences IS26 and IS5 were found downstream from bla(SHV-5). The DNA sequences of the genetic environment surrounding bla(SHV-5) were homologous to plasmid p1658/97 from Escherichia coli, containing a truncated recF gene and a truncated deoR gene upstream and downstream from bla(SHV-5), respectively. RecF may be involved in bla(SHV-5) translocation to the plasmid by RecF-dependent recombination. This novel genetic environment may be associated with the successful proliferation and/or expression of SHV-5 in K. pneumoniae strains from Taiwan.     

Diversity of bla genes and low incidence of CTX‐M in plasmid‐mediated AmpC‐producing Escherichia coli clinical isolates

The aim of this study was to characterize plasmid‐mediated AmpC (pAmpC)‐producing Escherichia coli clinical isolates. A total of 101 strains with AmpC‐susceptibility pattern were prospectively included. All isolates were tested by multiplex PCR to detect different bla genes. Phylogenetic groups were determined by a multiplex PCR assay. Antimicrobial susceptibility was tested by a microdilution commercial method. Presence of blapAmpC was detected in 79 (78.2%) of the strains; in these pAmpC‐producing isolates, blaTEM was detected in 41 (51.9%) strains, blaSHV in 5 (6.3%) strains, blaOXA in 3 (3.8%) strains, and blaCTX‐M in 3 (3.8%) strains. blaVIM and blaKPC were detected in one strain. Sixteen strains belonged to phylogroup A, 27 to B1, 20 to B2, and 16 to D. As conclusion, the majority of the strains of E. coli with AmpC‐susceptibility pattern are pAmpC positive, although the association of extended‐spectrum beta‐lactamases (ESBL) and pAmpC is unusual.     

Development of extended-spectrum activity in TEM β-lactamases in hyper-mutable, mutS Escherichia coli

TEM-1 and TEM(pUC19)beta-lactamases can gain activity against ceftazidime and other expanded-spectrum cephalosporins via point mutation. The frequency of emergent resistance to ceftazidime at 4 x MIC was elevated >or= 250-fold in hyper-mutable, MutS-deficient Escherichia coli harbouring these beta-lactamase genes on high- or low-copy plasmids. Moreover, although ceftazidime-resistant mutants, or those with reduced susceptibility, were selected in both the wild-type and mutS hosts, many more mutants in the mutS host showed ceftazidimase-type extended-spectrum beta-lactamase (ESBL) activity. This correlated with a G-A point mutation at position 484 in the bla(TEM-1) and bla(TEM-pUC19) genes, conferring the Arg164His amino-acid substitution found in the TEM-29 ESBL. Non-ESBL mutants lacked changes in bla(TEM).