vanM,a New Glycopeptide Resistance Gene Cluster Found in Enterococcus faecium

Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 μg/ml) and teicoplanin (MIC, 96 μg/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-d-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in d-Ala-d-Lac.Glycopeptide-resistant enterococci have emerged as important nosocomial pathogens since the late 1980s (18, 25). The glycopeptides vancomycin and teicoplanin act by binding to the d-alanyl-d-alanine (d-Ala-d-Ala) terminus of intermediates in peptidoglycan formation, inhibiting cell wall cross-linking (8). Resistance to glycopeptide antibiotics in enterococci results from the synthesis of peptidoglycan precursors with low affinity for these antibiotics, mediated by different van gene clusters (4, 8). Seven types of gene clusters conferring glycopeptide resistance have been described in enterococci based on DNA sequence and organization. They are designated according to the name of the ligase gene, which encodes either a d-Ala:d-Lac (vanA, vanB, and vanD) or a d-Ala:d-Ser (vanC, vanE, vanG, and vanL) ligase for the synthesis of peptidoglycan precursors with low affinity for glycopeptides. The vanA, vanB, and vanD gene clusters contain genes for a two-component regulatory system (vanR and vanS), three resistance genes (vanH, encoding dehydrogenase; vanA, vanB, or vanD, encoding ligase; vanX, encoding dd-dipeptidase); an accessory gene (vanY); and the vanZ gene, which is present in the vanA gene cluster, whereas the vanW gene is found only in the vanB operon (8). Another van gene cluster, vanF, has been described in a biopesticide, Paenibacillus popilliae (20), but has not yet been found in enterococci.Although vanA, vanB, and vanD gene clusters involve genes encoding similar enzymatic functions, they can be distinguished on the basis of the level and inducibility of resistance to glycopeptides and by the location of the genes (8, 11). The VanA type is characterized by acquired resistance to high levels of both vancomycin and teicoplanin, and it is induced by either vancomycin or teicoplanin. The VanB type is characterized by acquired resistance to various concentrations of vancomycin but not to teicoplanin and is induced only by vancomycin (1). VanA- and VanB-type resistances are the most common glycopeptide resistance phenotypes (23). The genes encoding the VanA and VanB phenotypes are carried on transposons, which may be found on plasmids or inserted into the chromosome (2).The VanD type is characterized by resistance to intermediate levels of vancomycin and to low levels of teicoplanin and is expressed constitutively (10, 12, 21). vanD genes appear to be located on the chromosome and are not transferable to other enterococci (12). This could explain the scarcity of recognized VanD strains in contrast to the widespread and high prevalence of VanA and VanB strains.Glycopeptide-resistant enterococci (GRE) were rare in China a few years ago (22), although vancomycin has been used in clinical practice for multidrug-resistant Gram-positive infections in mainland China for decades. In recent years, GRE strains isolated from hospitalized patients in mainland China have been increasing (6, 22, 27). In the course of determining the genotypes of glycopeptide-resistant Enterococcus faecium strains isolated from a teaching hospital in Shanghai, we found that several strains were negative by PCR for vanA, vanB, and vanD genes. The responsible glycopeptide resistance gene was cloned and sequenced from strain Efm-HS0661 and found to be novel. This new glycopeptide resistance gene cluster has been termed the vanM cluster. Of 10 unique GRE clinical strains isolated at our teaching hospital from 2005 to 2008, 6 carried vanM, which plays a role in the increasing GRE prevalence in China.     

Infections with Rhodococcus equi in children

Three cases of serious infection in children, including the first two reports of bacteremia, due to Rhodococcus equi are described. Only seven pediatric cases have been reported to date. In the laboratory, R. equi can easily be misidentified as a nonpathogenic Corynebacterium spp. (diphtheroid) or a Mycobacterium spp. Despite an overall mortality rate of 25% in adults, no pediatric deaths have occurred due to R. equi. The organism is generally susceptible to vancomycin, aminoglycosides, chloramphenicol, and erythromycin, but, optimal duration of antibiotic therapy is unknown. Treatment with multiple antibiotics was successful in all three cases reported here. Although only a small number of cases have been reported in children, R. equi appears to be an important pediatric pathogen.     

Comparison of Glycopeptide-Resistant Enterococcus faecium Isolates and Glycopeptide Resistance Genes of Human and Animal Origins

One hundred thirty-two glycopeptide-resistant Enterococcus faecium (GREF) isolates from different hospitals and pig and poultry farms in Belgium were compared on the basis of (i) their antibiotic susceptibilities, (ii) their SmaI pulsed-field gel electrophoresis (PFGE) patterns, and (iii) the organization of their Tn1546 or related elements in order to detect possible phenotypic and genotypic relationships among both groups of isolates. Human and animal vanA-positive GREF isolates were found to have similar susceptibility patterns; they remained susceptible to gentamicin and were, in general, susceptible to ampicillin. PFGE demonstrated a very high degree of genomic heterogeneity in both groups of isolates. However, indistinguishable isolates were found within different farms or hospitals, and in two instances, epidemiologically unrelated pig and human isolates showed indistinguishable PFGE patterns. In total, eight different transposon types were identified, and all were related to the prototype transposon Tn1546. The two predominant types, Tn1546 and type 2 transposons, which differed at three band positions, were present in both human and animal isolates. Type 2 transposons were significantly associated with pig isolates. The other types were seldom detected. These data suggest a possible exchange of glycopeptide resistance markers between animals and humans.     

Transferable, Plasmid-Mediated VanB-Type Glycopeptide Resistance in Enterococcus faecium

An approximately 60-kb transferable, vanB-carrying plasmid has been identified in a clinical Enterococcus faecium strain. A similar plasmid has been observed in an unrelated E. faecium strain, suggesting that plasmid transfer of vanB operons occurs in nature and plays a role in the dissemination of VanB-type resistance among strains of E. faecium.     

VapB-positive Rhodococcus equi infection in an HIV-infected patient in Japan

Rhodococcus equi, a bacterium present in soil, is a common cause of pneumonia in foals. This organism has been recognized as an opportunistic pathogen in humans, typically causing infection in immunocompromised hosts such as HIV-infected patients and organ transplant recipients. However, human infection with R. equi has not been reported in Japan except in a case involving a laboratory worker. We report the first human case of VapB-positive R. equi pneumonia, which involved an HIV-infected patient living in an urban area in Japan. The patient was treated successfully with 450mg rifampicin and 600mg tosufloxacin, even though his CD4+ lymphocyte count at the time of diagnosis was 10/µl. The patients dogs were suspected in the epidemiology of this infection, but unfortunately we could not isolate the organism from canine-associated specimens in this case. R. equi infections in companion animals have been thought to be very rare, but they may be increasing in dogs. Therefore, further epidemiological research may clarify the prevalence of R. equi infection and the factors predisposing dogs to this infection.     

In Vitro Activity of the New Glycopeptide LY333328 against Multiply Resistant Gram-Positive Clinical Isolates

The in vitro activity of LY333328 was compared with those of vancomycin and teicoplanin against 425 gram-positive clinical isolates, including a variety of multiply resistant strains. LY333328 at ≤4 μg/ml inhibited all microorganisms tested, including methicillin- and teicoplanin-resistant staphylococci, glycopeptide-resistant enterococci, penicillin- and multiply resistant pneumococci, and viridans and beta-hemolytic streptococci.     

Therapy of Rhodococcus equi disseminated infections in nude mice.

Rhodococcus equi is a facultative, intracellular, gram-positive coccobacillus increasingly reported as an opportunistic pathogen in human immunodeficiency virus-positive patients. However, the optimal drug regimen for treating R. equi pulmonary or systemic infections is not yet known. Therefore, a model of intravenously infected nude mice with disseminated infection was created to study the efficacy of antibiotics alone or in combination as determined by the reduction of bacterial CFU per gram in the lungs and spleen after 4 and 11 days of treatment. The studied antibiotics possessing low MICs against R. equi strains were amikacin, ciprofloxacin, erythromycin, imipenem, minocycline, rifampin, and vancomycin. Vancomycin, imipenem, and rifampin were the most effective agents in monotherapy. On the other hand, amikacin, ciprofloxacin, erythromycin, and minocycline alone were not active in this model. The most active drug combinations were those including vancomycin. No antibiotic-resistant mutants were selected in vivo with treatment involving any drugs used alone or in combination. Although the treatment recommended until now for R. equi infections is rifampin plus erythromycin, this study suggests that antibiotic combinations which include vancomycin may be the most effective in vivo.     

Relapsing Rhodococcus equi infection in a heart transplant recipient successfully treated with long-term linezolid

We report a heart transplant recipient with Rhodococcus equi and Cryptococcus neoformans infection. Despite an initially relapsing course and the acquisition of resistance in vivo, the patient responded to 5 months of linezolid and 6 months of fluconazole. This is the 1st R. equi infection to be cured with linezolid in a heart transplant recipient.     

Penicillin-binding proteins of Rhodococcus equi: potential role in resistance to imipenem.

Rhodococcus equi is a gram-positive coccobacillus which, like other members of the order Actinomycetales, is increasingly reported as an opportunistic pathogen in patients with AIDS. The use of combinations of antibiotics that include imipenem (IMP) has been suggested for the treatment of patients infected with R. equi. An antagonism between IMP, meropenem, cefoxitin, ceftriaxone, moxalactam, and oxacillin and other beta-lactams, such as penicillin, amoxicillin, cephalothin, and ticarcillin, was detected in vitro both on Mueller-Hinton agar and in broth for all 10 IMP-susceptible R. equi strains examined. To study the mechanism of the antagonism between beta-lactams, a mutant with decreased susceptibility to IMP (isolate IpR) was selected in vitro from a susceptible clinical isolate of R. equi (isolate IpS). IpR exhibited decreased susceptibility to IMP, meropenem, cefoxitin, ceftriaxone, moxalactam, and oxacillin but not to penicillin, amoxicillin, cephalothin, or ticarcillin. No beta-lactamase was found in IpS, IpS cultured with antagonistic beta-lactams, or IpR strains. Labeling of penicillin-binding proteins (PBPs) revealed four PBPs with molecular masses of ca. 59, 56, 43, and 26 kDa in IpS. In IpR, PBP 3 disappeared and was replaced by PBP 3a of 40 kDa. The 50% saturation of PBP 3 and PBP 3a by the carbapenems correlated with the MICs of these antibiotics, respectively, for IpS and IpR strains. However, PBP 3a was not detected in IpS when IpS was cultured in the presence of beta-lactams, with which antagonism was observed. The present work describes the PBPs of R. equi and reports that IMP resistance in R. equi is related to an altered PBP pattern.     

艾滋病并发肺部马红球菌感染的鉴定及药敏分析

目的 探讨艾滋病患者肺部感染马红球菌的生物学性状及耐药情况.方法 取艾滋病患者痰液、血液、经皮肺穿刺物、纤维支气管镜肺泡灌洗液做细菌培养鉴定及药物敏感实验,并对结果进行分析.结果 16株马红球菌为多形态革兰阳性杆菌,均产生橙黄、橘红色素,需氧、无芽孢、无动力、有荚膜;不发酵任何糖类、醇类,触酶、硝酸盐、α-葡萄苷酶呈阳性反应.对环丙沙星和头孢哌酮的敏感率为100.0%,其他药物敏感率依次为氧氟沙星和利福平(87.5%),头孢噻嗪和氯霉素(75.0%);头孢哌酮、氧氟沙星和利福平耐药率为0.0%,环丙沙星、头孢噻嗪耐药率为6.25%,氯霉素为12.5%.结论 马红球菌为人类机会致病菌,掌握其生物学性状及药敏规律,对识别和治疗艾滋病肺部感染患者及指导临床合理用药具有重要的临床价值.     

The msaABCR Operon Regulates Resistance in Vancomycin-Intermediate Staphylococcus aureus Strains

Vancomycin-intermediate Staphylococcus aureus (VISA) strains present an increasingly difficult problem in terms of public health. However, the molecular mechanism for this resistance is not yet understood. In this study, we define the role of the msaABCR operon in vancomycin resistance in three clinical VISA strains, i.e., Mu50, HIP6297, and LIM2. Deletion of the msaABCR operon resulted in significant decreases in the vancomycin MIC (from 6.25 to 1.56 μg/ml) and significant reductions of cell wall thickness in strains Mu50 and HIP6297. Growth of the mutants in medium containing vancomycin at concentrations greater than 2 μg/ml resulted in decreases in the growth rate, compared with the wild-type strains. Mutation of the msaABCR operon also reduced the binding capacity for vancomycin. We conclude that the msaABCR operon contributes to resistance to vancomycin and cell wall synthesis in S. aureus.     

红霉素耐药葡萄球菌对克林霉素诱导性耐药的研究

目的：了解临床分离的红霉素耐药葡萄球菌对克林霉素诱导性耐药的发生率,比较双纸片扩散法（D试验）中纸片之间距离对试验结果的影响。方法VITEK-2微生物分析系统检测为红霉素耐药、克林霉素敏感或中介的葡萄球菌135株,其中金黄色葡萄球菌（SA）为55株,凝固酶阴性葡萄球菌（CNS）为80株。比较红霉素纸片与克林霉素纸片边距15 mm与26 mm对结果的影响。结果 D试验阳性率为57.0%,其中SA阳性率70.9%, CNS为47.5%（P＜0.01）。纸片边距15 mm组D试验阳性77株,26 mm组仅69株,漏检率10.4%（P＜0.05）。结论实验室应常规开展D试验,D试验中双纸片间距以15 mm为佳。     

Monooxygenase-like sequence of a Rhodococcus equi gene conferring increased resistance to rifampin by inactivating this antibiotic.

A DNA clone from Rhodococcus equi conferring low-level rifampin resistance through the ability to inactivate this antibiotic via its decomposition was identified. The iri (inactivation of rifampin) gene consisted of an open reading frame of 1,437 bp encoding a 479-amino-acid sequence strongly resembling those of monooxygenases acting upon phenolic compounds or involved in polyketide antibiotic synthesis. When expressed in Escherichia coli, the gene conferred resistance to a > 50-micrograms/ml concentration of the drug.     

Activity of Clarithromycin or Rifampin Alone or in Combination against Experimental Rhodococcus equi Infection in Mice

Treatment of mice with the combination of clarithromycin with rifampin resulted in a significantly lower number of Rhodococcus equi CFU in the organs of mice than treatment with either drug alone or placebo. There was no significant difference in the number of R. equi CFU between mice treated with clarithromycin monotherapy, rifampin monotherapy, or placebo. The combination of clarithromycin with rifampin conferred a clear advantage over either drug as monotherapy in this model of chronic R. equi infection.     

In vitro susceptibilities of Rhodococcus equi and other common equine pathogens to azithromycin,clarithromycin, and 20 other antimicrobials

The objective of this study was to determine in vitro activities of azithromycin (AZM), clarithromycin (CLR), and 20 other antimicrobial agents against Rhodococcus equi and other common equine bacterial pathogens. A total of 201 bacterial isolates from various equine clinical samples were examined. CLR was more active than AZM against R. equi, with MICs at which 90% of the isolates were inhibited of 0.12 and 1.0 micro g/ml, respectively. Other antimicrobial agents highly active against at least 90% of R. equi isolates in vitro included rifampin, gentamicin, and imipenem. Both AZM and CLR showed good activity against beta-hemolytic streptococci and Staphylococcus spp. AZM was more active than other macrolides against Pasteurella spp. and Salmonella enterica.     

Environmental Isolates of Azole-Resistant Aspergillus fumigatus in Germany

Azole antifungal drug resistance in Aspergillus fumigatus is an emerging problem in several parts of the world. Here we investigated the distribution of such strains in soils from Germany. At a general positivity rate of 12%, most prevalently, we found strains with the TR₃₄/L98H and TR₄₆/Y121F/T289A alleles, dispersed along a corridor across northern Germany. Comparison of the distributions of resistance alleles and genotypes between environment and clinical samples suggests the presence of local clinical clusters.