Detection of Methicillin Resistance in Coagulase-Negative Staphylococci Initially Reported as Methicillin Susceptible Using Automated Methods

Reliable detection of methicillin resistance in coagulase-negative staphylococci (CNS) is required for appropriate therapy of serious infections from these pathogens. To determine the most accurate method of measuring methicillin resistance in CNS initially reported as methicillin susceptible by automated methods, we compared mecA detection by polymerase chain reaction (PCR) with phenotypic methods. One hundred eighty-eight blood culture isolates of CNS that were initially reported as susceptible to methicillin using commercial methods (Vitek or MicroScan) were tested by agar dilution, disk diffusion, oxacillin salt agar screen plate, and a multiplex PCR assay using primer sets for mecA and 16S rRNA. Sixteen isolates (8.5%) previously reported as methicillin susceptible by automated methods contained the mecA gene. MICs of these isolates ranged from 0.5 μg/mL to ≥128 μg/mL. Ten of these isolates had MICs equal to or below the NCCLS breakpoint of 2 μg/mL. Six of the 10 isolates (4 with MICs of 0.5 μg/mL and 2 with MICs of 2 μg/mL) did not grow on any of the oxacillin screen plates after 48 h of incubation at 30°C or 35°C. All six isolates were induced to grow in the presence of oxacillin at 128 μg/mL by serial passaging on plates containing increasing concentrations of antibiotic. Retesting with MicroScan and Vitek detected methicillin resistance in 7 and 10 isolates, respectively. Disk diffusion testing with incubation for 48 h proved to be the next best method after PCR for detection of methicillin resistance (15 of 16 isolates). Commercial automated methods and some methods recommended by National Committee for Clinical Laboratory Standards may not detect methicillin resistance in CNS that carry the mecA gene and have MICs just below breakpoint.     

Distribution of Insertion Sequence-Like Element IS1272 and Its Position Relative to Methicillin Resistance Genes in Clinically Important Staphylococci

The distribution of insertion sequence-like element IS1272 was analyzed for clinical isolates of Staphylococcus aureus, Staphylococcus epidermidis, and Staphylococcus haemolyticus. In each of the staphylococcal species, IS1272 was detected in both methicillin-resistant (MR) and methicillin-susceptible strains of different genetic types. In MR isolates, IS1272 was generally located downstream of the truncated mecR1 gene (DeltamecR1), with an identical junction sequence occurring between DeltamecR1 and IS1272, although insertion of an additional gene sequence in the junction sequence was detected in one S. epidermidis isolate. These findings suggest that the mec element with the rearranged form of mecR1 (DeltamecR1-IS1272) has been transmitted to multiple clones of staphylococci.     

Mutations in Ribosomal Protein L3 Are Associated with Oxazolidinone Resistance in Staphylococci of Clinical Origin

Following recent reports of ribosomal protein L3 mutations in laboratory-derived linezolid-resistant (LZD^r) Staphylococcus aureus, we investigated whether similar mutations were present in LZD^r staphylococci of clinical origin. Sequence analysis of a variety of LZD^r isolates revealed two L3 mutations, ΔSer145 (S. aureus NRS127) and Ala157Arg (Staphylococcus epidermidis 1653059), both occurring proximal to the oxazolidinone binding site in the peptidyl transferase center. The oxazolidinone torezolid maintained a ≥8-fold potency advantage over linezolid for both strains.Oxazolidinone resistance in clinical staphylococci is most often associated with mutations in 23S rRNA domain V, in particular G2576T (Escherichia coli numbering) (24, 30). Other 23S rRNA mutations, such as G2447T, until recently (19) were strictly associated with laboratory-derived strains (28). Methylation of 23S rRNA (A2503) by the horizontally transmitted Cfr methyltransferase also confers resistance to linezolid (LZD) as well as phenicols, lincosamides, pleuromutilins, and streptogramin A (12, 29). Incidences of LZD resistance in strains lacking 23S rRNA mutations or the cfr gene have prompted analysis of other structural components of the ribosome which may have the potential to influence oxazolidinone binding.A number of 50S large-subunit ribosomal proteins have regions which interact closely with the oxazolidinone binding site in the peptidyl transferase center (PTC), most notably L3 and L4. In rare cases, mutations in L4 have been implicated in LZD nonsusceptibility in clinical Streptococcus pneumoniae isolates (32) and in laboratory-derived Staphylococcus aureus strains (17). Mutations in L3 have typically been associated with resistance to pleuromutilins (whose binding site overlaps with that of oxazolidinones in the PTC) such as tiamulin (TIA) and retapamulin (1, 2, 8, 13, 20, 22). However, we recently described a variety of L3 mutations in S. aureus following in vitro selection with oxazolidinones LZD and torezolid (TR-700) (17) a novel oxazolidinone with enhanced potency against a broad range of gram-positive pathogens, including strains resistant to LZD (11, 14, 26, 27).To investigate the relevance of L3 mutations to clinical oxazolidinone resistance, we sequenced L3-encoding rplC genes in 11 Lzd^r clinical isolates, 2 of which included the uncharacterized Staphylococcus epidermidis strain 1653059 (cfr negative; methicillin [meticillin]-resistant S. epidermidis [MRSE]; Eurofins Medinet, Inc., Chantilly, VA) and S. aureus strain NRS127 (cfr negative; methicillin resistant; Network of Antimicrobial Resistance in Staphylococcus aureus [NARSA] collection, Chantilly, VA), previously reported as having an unknown, non-23S rRNA-based resistance mechanism (27).(Portions of this work were presented at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy [16], San Francisco, CA, 12 to 15 September 2009.)Chromosomal DNA was isolated, and PCR amplification of the six S. aureus rrn alleles was performed as previously described (17, 21). The 3′ portions of S. epidermidis 23S rRNA genes were amplified using the VdomainF primer in conjunction with 23S rRNA allele-specific downstream flanking reverse primers (Se_rrlA-FR) designed using the S. epidermidis RP62A genome sequence (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_002976","term_id":"57865352","term_text":"NC_002976"}}NC_002976) (Table ​(Table1).1). Genes encoding ribosomal proteins L3 (rplC), L4 (rplD), and L22 (rplV) were amplified as a single amplicon (∼3.3 kb) from S. aureus (rplCF/rplVR) (17) and S. epidermidis (rplCF/Se_rplVR) (Table ​(Table1).1). Sequencing of PCR products (Retrogen, Inc., San Diego, CA) was performed with primers flanking the 23S rRNA domain V region (21) and individual ribosomal protein genes (Table ​(Table11).

TABLE 1.

Primers used to amplify and sequence 23S rRNA and ribosomal protein genes

Lividomycin Resistance in Staphylococci by Enzymatic Phosphorylation

Enzymatic inactivation of lividomycin (LV) was attempted with nine clinical isolates of staphylococci including LV-susceptible and -resistant strains. LV inactivation and the incorporation into LV of (32)P from gamma-(32)P-adenosine triphosphate were demonstrated in the presence of cell-free extracts from LV-resistant strains but not from LV-susceptible ones. The enzyme was purified approximately 82-fold from a resistant Staphylococcus aureus strain by means of ammonium sulfate fractionation and column chromatography. Some properties of the partially purified LV-phosphorylating enzyme were quite similar to those of an enzyme from Escherichia coli carrying an R factor conferring LV resistance, and the phosphorylated product of the drug was also found to be identical with that produced by E. coli carrying an R factor, i.e., 5'-phosphoryl-LV.     

First Report of Plasmid-Mediated Quinolone Resistance Determinant qnrS1 in an Escherichia coli Strain of Animal Origin in Italy

A qnrS1-positive strain of Escherichia coli was detected among 73 poultry isolates showing ciprofloxacin MICs of ≥0.125 μg/ml. The qnrS1 gene was associated with a Tn3-like transposon, as previously described to occur in a Salmonella enterica serovar Infantis strain of animal origin, but the plasmid scaffold carrying this element resembled that of a plasmid previously identified in Salmonella enterica serovar Dublin. These elements suggest genetic exchanges among Salmonella and E. coli and a potential animal reservoir for the qnr genes.Three plasmid-mediated quinolone resistance mechanisms have been described so far: Qnr peptides, capable of protecting DNA gyrase and topoisomerase IV from quinolones; Aac(6′)-Ib-cr aminoglycoside acetyltransferase, modifying the quinolones with a piperazinyl substituent (e.g., ciprofloxacin); and the quinolone efflux pump QepA. Plasmid-mediated quinolone resistance is being increasingly recognized in Enterobacteriaceae from human infections but seems very rare in strains of animal origin (13). However, in a recent study from China, 16.9% of the isolates from food-producing animals contained one or more plasmid-mediated quinolone resistance determinants (10). In Europe, qnr-carrying Escherichia coli strains have not yet been described to occur in animals, and Qnr peptides have been reported to occur only in Salmonella enterica serovar Infantis isolates from chicken carcasses in Germany and in Salmonella enterica serovar Bredeney isolates from chicken meat in The Netherlands (9, 14). In this study, the occurrences of qnr, aac(6′)-Ib-cr, and qepA genes in 73 E. coli strains of avian origin were investigated. These 73 strains were all those showing ciprofloxacin MICs of ≥0.125 μg/ml among 113 isolates recovered between April 2003 and December 2006 (18, 25, 27, and 43 isolates collected in 2003, 2004, 2005, and 2006, respectively) during the surveillance activities of the Istituto Zooprofilattico delle Venezie (Legnaro, Italy). The 113 isolates (74 from poultry with colibacillosis and 39 from poultry at slaughter) represented over 10% of all the E. coli isolates from poultry collected during the study period in the Italian region which hosts the greatest number of poultry farms. Of the 73 isolates analyzed, 65 were fully resistant to ciprofloxacin (MIC range, 4 to 32 μg/ml) and 8 showed reduced susceptibility (MIC range, 0.125 to 0.5 μg/ml). MICs were determined by using Etest kits (AB Biodisk, Solna, Sweden) in accordance with the manufacturer''s recommendations; the interpretative breakpoints were based on CLSI susceptibility criteria (6, 7). The screening for the qnrA, qnrB, qnrS, aac(6′)-Ib, and qepA genes was carried out by multiplex and simplex PCR amplifications, using primers and conditions previously described (2, 12, 15), and amplicons were sequenced to determine the gene variants. One qnrS1-positive isolate (strain 3963) was detected among the eight isolates showing reduced susceptibility to ciprofloxacin (12.5%); all the other isolates of this collection were negative for the qnr, aac(6′)-Ib, and qepA genes. Strain 3963 was isolated from a regularly slaughtered chicken in 2006. This strain belonged to phylogenetic group D (5) and to multilocus sequence type 398 (http://mlst.ucc.ie/mlst/dbs/Ecoli). Strain 3963 showed resistance to enrofloxacin and reduced susceptibility to nalidixic acid, ciprofloxacin, and levofloxacin (Table ​(Table1)1) (according to references 6 and 7). This strain was also resistant to ampicillin but susceptible to broad-spectrum cephalosporins. No mutations were identified in the quinolone resistance-determining regions of the gyrA, gyrB, and parC genes (16). Strain 3963 also carried the bla_TEM-1 gene, as demonstrated by PCR and sequencing using primers and conditions previously described (11).

TABLE 1.

Susceptibilities of the qnrS1 donor (3963), transformant (TOP10-3963), and recipient (E. coli TOP10) strains to selected antibiotics

Evolution and Dissemination of OqxAB-Like Efflux Pumps,an Emerging Quinolone Resistance Determinant among Members of Enterobacteriaceae

The OqxAB efflux pump, a plasmid-mediated quinolone resistance (PMQR) determinant, has become increasingly prevalent among members of Enterobacteriaceae over the past decade. To investigate the evolution and dissemination routes of the oqxAB operon, we assessed the prevalence of oqxAB-like elements among various Gram-negative bacterial species and analyzed the genotypic and phenotypic characteristics of organisms harboring such elements. With a comprehensive genotyping approach, a chromosome-based oqxAB operon was detectable in all Klebsiella pneumoniae strains tested, including organisms isolated before the year 1984. Sequence and phylogenetic analyses confirmed that the oqxAB operon in K. pneumoniae isolates was genetically closest to their plasmid-borne counterparts recoverable only from Escherichia coli and Salmonella isolates collected from the year 2003 onward. Chromosomal elements with much lower sequence homology were also found among the Enterobacter spp. but not other Gram-negative species. Contrary to the quinolone resistance phenotypes which were consistently observable among organisms with oqxAB-harboring plasmids, chromosomal oqxAB elements generally did not confer quinolone resistance, except for K. pneumoniae strains, which exhibited a typical oqxAB-mediated phenotype characterized by cross-resistance to olaquindox, chloramphenicol, and the quinolones. Gene expression analysis illustrated that such phenotypes were due to elevated expression of the chromosomal oqxAB operon. Furthermore, transposition of the oqxAB operon from the bacterial chromosome to plasmids was found to result in a >80-fold increase in the level of expression of the OqxAB pump, confirming its status as the first constitutively expressed efflux system located in bacterial mobile elements.     

Suppression of Intrinsic Resistance to Methicillin and Other Penicillins in Staphylococcus aureus

The pH of the medium in which staphylococcal susceptibility to penicillins was determined was found to make a profound difference (128- to 8,000-fold) in the expression of "intrinsic" resistance, whereas beta-lactamase-mediated resistance was only slightly affected by pH; methicillin-resistant staphylococci that are beta-lactamase-negative are models of pure intrinsic resistance, and the common beta-lactamase-producing organisms (methicillin-susceptible) are examples of pure beta-lactamase-mediated resistance. Methicillin-resistant staphylococci were unable to express their resistance at pH 5.2. However, growth of methicillin-resistant organisms in acid (pH 5.2) medium, followed by susceptibility testing at pH 7.4, showed no elimination of the genotype for intrinsic resistance, indicating that the pH effect was due to suppression, rather than to elimination of the gene determining the intrinsic resistance. These pH changes had little effect on the susceptibility of staphylococci that possessed neither intrinsic resistance nor beta-lactamase-mediated resistance. Thus, the suppression of "intrinsic" resistance was highly specific, and probably not the result of a change in ionization of the antibiotic, which would have been expected to affect all cells essentially equally. It is unlikely that foci of inflammation in man become sufficiently acid to suppress methicillin resistance of the staphylococci causing infection and inflammation.     

Genotypic Stability of Methicillin Resistance in Staphylococcus aureus at Supraoptimal Temperature

Methicillin resistance in a highly resistant mutant of Staphylococcus aureus could not be diminished by elevated temperature. After 100 subcultures at 43 degrees C the resistance to methicillin was retained when determined at 37 degrees C.     

Molecular Cloning and Functional Analysis of a Novel Tetracycline Resistance Determinant, tet(V), from Mycobacterium smegmatis

The nucleotide sequence and mechanism of action of a tetracycline resistance gene from Mycobacterium smegmatis were determined. Analysis of a 2.2-kb sequence fragment showed the presence of one open reading frame, designated tet(V), encoding a 419-amino-acid protein (molecular weight, 44,610) with at least 10 transmembrane domains. A database search showed that the gene is homologous to membrane-associated antibiotic efflux pump proteins but not to any known tetracycline efflux pumps. The steady-state accumulation level of tetracycline by M. smegmatis harboring a plasmid carrying the tet(V) gene was about fourfold lower than that of the parental strain. Furthermore, the energy uncoupler carbonyl cyanide m-chlorophenylhydrazone blocked tetracycline efflux in deenergized cells. These results suggest that the tet(V) gene codes for a drug antiporter which uses the proton motive force for the active efflux of tetracycline. By primer-specific amplification the gene appears to be restricted to M. smegmatis and M. fortuitum.     

ICU耐甲氧西林金黄色葡萄球菌耐药性监测及利奈唑胺治疗效果的研究

目的:通过对重症监护病房(ICU)耐甲氧西林金黄色葡萄球菌(MRSA)感染的分析,探讨其耐药性;并观察利奈唑胺在MRSA感染中的治疗效果.方法:收集我院ICU 2007年1月～2010年1月入住ICU患者的痰、血、尿、脑脊液、中心静脉穿刺针顶端及局部引流物进行细菌学培养及药物敏感性试验.结果:我院ICU病房3年共发生MRSA 54例,绝大多数为耐药菌,万古霉素、替考拉宁和利奈唑胺对MRSA感染的敏感率均为100%.利奈唑胺组的有效率和痊愈率为78.9%和36.8%.结论:控制MRSA的感染应积极进行病原学监测、及时发现病例、合理使用广谱抗生素.利奈唑胺是MRSA感染的有效药物,且不良反应较少.     

Genomic Epidemiology of Klebsiella pneumoniae in Italy and Novel Insights into the Origin and Global Evolution of Its Resistance to Carbapenem Antibiotics

Klebsiella pneumoniae is at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniae worldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.     

耐甲氧西林金黄色葡萄球菌耐药机制研究进展

金黄色葡萄菌是临床最常见的致病菌之一，在临床致病菌中分离率位居前列，其中以耐甲氧西林金黄色葡萄球菌（MRSA）尤为重要，由于其多重耐药性和易造成医院内感染的暴发流利，已成为临床抗感染治疗的难点。随着抗生素种类不断增加及临床上的广泛应用和MRSA多重性耐药性，全身感染的病死率高达50％以上。文献报道1993年日本／cansai医大附属医院MRSA分离率为41．0％，1999年英国30个临测中心MRSA检出率高达56．7％，1999年美国疾病控制中心（CDC）统讣全美重症监护病房内感染患者MRSA阳性率占53．2％。我国MRSA的感染率也相当高，李争鸣等研究报道MRSA检出率达52．34％，张银旺报道的MRSA检出率基本相同，达52．4％。     

Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the qacA Locus

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing the qacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in the qacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance gene qacA also mediates in vitro resistance to cationic tPMP-1.     

梅里埃VTIEK MS 质谱仪在甲氧西林耐药及敏感的金黄色葡萄球菌快速鉴定的应用研究

目的　探讨梅里埃VTIEK MS 质谱仪快速鉴定甲氧西林耐药金黄色葡萄球菌（MRSA）及甲氧西林敏感的金 黄色葡萄球菌(MSSA) 的可行性。方法　对广州市番禺区中心医院2018 ～ 2019 年各个病区住院患者在不同部位分离的 30 株MRSA 及20 株MSSA,用VITEK 2 Compact 的药敏卡AST-P639 进行耐药性检测,用VTIEK MS 对所有菌株进 行鉴定。结果　用VITEK MS 的Launch pad 软件对30 株MRSA 及20 株MSSA 所产生的质谱峰图m/z 进行分析,质谱 峰m/z 的 3 009,5 624 和6 887 为金黄色葡萄球菌的特征峰,质谱峰m/z 2 637 及4 308 为区分MRSA 及MSSA 最主要 的特征峰,其中质谱峰m/z 2 637 MSSA 组较MRSA 组低,质谱峰m/z 4 308 则MRSA 组较MSSA 组高,不同菌株质谱 峰略有不同。结论　梅里埃VTIEK MS 可快速准确鉴别MRSA 及MSSA,且具有耗时短、灵敏度高、特异性好等特点。