A 12 year (1998-2009) antibiotic resistance surveillance of Klebsiella pneumoniae collected from intensive care and urology patients in 14 Dutch hospitals.

OBJECTIVES: We evaluated the changes in antibiotic resistance from 1998 to 2009 of Klebsiella pneumoniae isolated from the intensive care units (ICUs) and urology services of 14 Dutch hospitals and the consequences for empirical therapy. METHODS: Quantitative antibiotic susceptibility testing of K. pneumoniae was performed in a central laboratory using a microbroth dilution method. Breakpoints were as defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The prevalence of extended-spectrum ß-lactamase (ESBL)- and carbapenemase-producing isolates was determined. RESULTS: A significant increase in resistance among ICU isolates was observed for ceftazidime (4.2%-10.8%), ciprofloxacin (5.8%-18.5%) and trimethoprim/sulfamethoxazole (11.9%-23.1%), and for cefuroxime (2.8%-7.9%) and trimethoprim/sulfamethoxazole (13.5%-27.8%) among urology isolates. Among ICU isolates the prevalence of ESBLs increased significantly from 2% to 8%. Carbapenemase production was not demonstrated. Among ICU isolates the prevalence of multidrug resistance increased and has been ≥12% since 2004. Among urology isolates multidrug resistance was highest in 2009 at 7.4%. Overall, resistance was significantly higher among ICU isolates. CONCLUSIONS: We observed an increase in resistance among ICU and urology isolates and an increased prevalence of ESBLs among ICU isolates. Carbapenemase production was not demonstrated. A regular update of empirical treatment protocols based on actual surveillance data is justified.

Diminished in vitro antibacterial activity of oxacillin against clinical isolates of borderline oxacillin-resistant Staphylococcus aureus.

Since it is unknown whether ß-lactam antimicrobial agents can be used effectively against borderline oxacillin-resistant Staphylococcus aureus (BORSA) with oxacillin MICs ≥4 mg/L, the in vitro bactericidal activity and pharmacodynamic effect of oxacillin against clinical BORSA isolates was evaluated. Time-kill experiments with oxacillin were performed and the results compared with those obtained with vancomycin, daptomycin and linezolid against BORSA with oxacillin MICs ≥4 mg/L and BORSA with oxacillin MICs ≤2 mg/L. Furthermore, the effect of ß-lactamase production and plasmid profile analysis were taken into account to clarify responses to oxacillin. Oxacillin killing activity was attenuated against BORSA compared with ATCC 29213 since the pharmacodynamic parameters revealed that the potency of oxacillin was markedly reduced (c. ten-fold) against BORSA with oxacillin MICs ≥4 mg/L. pBORa53-like plasmid-containing BORSA with oxacillin MICs ≤2 mg/L showed markedly more regrowth. In conclusion, oxacillin was non-effective in the eradication of either (i) BORSA with oxacillin MICs ≥4 mg/L or (ii) ß-lactamase-hyperproducing BORSA (MICs ≤2 mg/L). Further investigation into ß-lactam dosing strategies against different BORSA strains is warranted in order to avoid possible therapy failure.

Molecular typing of methicillin-susceptible Staphylococcus aureus isolates collected in the Yogyakarta area in Indonesia, 2006.

The characterization of 62 community-associated methicillin-sensitive Staphylococcus aureus (MSSA) isolates from 440 individuals in the Yogyakarta area of Indonesia in 2006 showed that: (i) almost half of the isolates were associated with methicillin-resistant S. aureus lineages [clonal complex (CC)1, CC8 and CC45] and (ii) ten Panton-Valentine leukocidin-positive isolates were associated with CC1 (n = 7), CC30 (n = 1) and CC51 (n = 2). The high Panton-Valentine leukocidin prevalence (16%) among S. aureus is of concern because these strains can cause severe infections and the introduction of staphylococcal cassette chromosome mec into virulent and epidemic MSSA could pose a serious public health threat.

Population structure of Staphylococcus aureus strains isolated from intensive care unit patients in the netherlands over an 11-year period (1996 to 2006).

The genetic background and the presence of several virulence factors of Staphylococcus aureus isolates from intensive care unit (ICU) patients from 14 hospitals in The Netherlands isolated from 1996 until 2006 were investigated. In total, 936 methicillin-susceptible S. aureus (MSSA) and 7 methicillin-resistant S. aureus (MRSA) isolates were collected. The genetic background was determined by spa typing and multilocus sequence typing (MLST). The virulence determinants Panton-Valentine leukocidin (PVL), toxic shock syndrome toxin 1 (TSST-1), and collagen adhesion (CNA) were detected with real-time PCR assays. On the MRSA isolates, mobile resistance staphylococcal cassette chromosome mec (SCCmec) typing was performed. Among the MSSA isolates, 313 different spa types were observed. A genetic background common to MRSA clones, e.g., MLST clonal complex 1 (CC1), CC5, CC8, CC22, CC30, and CC45, was observed among 62% of the isolates. The remaining isolates were associated with MSSA-related MLST CCs. MLST CC1, CC25, and CC30 were continuously present, and other MLST CCs fluctuated over time. Two percent of the MSSA isolates harbored PVL, 21% had TSST-1, and 46% were positive for CNA. There were no changes in the prevalence of the virulence factors over time. Four MRSA isolates were typed as ST8-MRSA-IV (where ST is the MLST sequence type and IV is the SCCmec type), two were ST5-MRSA-II, and one was ST228-MRSA-I. All MRSA isolates were PVL, CNA, and TSST-1 negative except for the two ST5-MRSA-II isolates, which were TSST-1 positive. No changes in the S. aureus genetic background and the prevalence of the virulence factors PVL, CNA, and TSST-1 were observed in ICU patients in The Netherlands over time.

Flucloxacillin, still the empirical choice for putative Staphylococcus aureus infections in intensive care units in the Netherlands.

OBJECTIVES: To determine the usefulness of flucloxacillin as empirical therapy for putative Staphylococcus aureus infections in intensive care unit (ICU) patients in the Netherlands, the antibiotic resistance of S. aureus isolates from ICUs over a 13 year period was investigated. METHODS: From 1996 to 2008, 1146 consecutive S. aureus isolates from ICU patients in 14 large referral hospitals were collected. The susceptibility to relevant antibiotics was determined by microbroth dilution according to CLSI guidelines. RESULTS: Resistance to flucloxacillin was only found in 12 isolates (1%). The resistance to clarithromycin, ciprofloxacin and moxifloxacin showed a significant trend over time, from 4.2% to 10.3%, from 1.0% to approximately 10% and from 0.0% to approximately 5.0%, respectively (P < 0.05). The resistance to penicillin, clindamycin and doxycycline increased over time, from 74% to 75%, from approximately 3.0% in 1996 to 3.2% in 2008 and from 2.2% in 1996 to 8.2% in 2008, respectively (P > 0.05). Resistance to cephalosporins, carbapenems, rifampicin and gentamicin was sporadically observed. No resistance was found to vancomycin, teicoplanin and linezolid. CONCLUSIONS: The empirical choice of flucloxacillin in the case of putative S. aureus infections in patients admitted to ICUs in the Netherlands is still justified.

Genetic diversity of methicillin-resistant Staphylococcus aureus in a tertiary hospital in The Netherlands between 2002 and 2006.

The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.

Chemokines and chemokine receptors encoded by cytomegaloviruses.

CMVs carry several genes that are homologous to genes of the host organism. These include genes homologous to those encoding chemokines (CKs) and G protein-coupled receptors (GPCRs). It is generally assumed that these CMV genes were hijacked from the host genome during the long co-evolution of virus and host. In light of the important function of the CK and GPCR families in the normal physiology of the host, it has previously been hypothesized that the CMV homologs of these proteins, CMV vCKs and vGPCRs, may also have a significant impact on this physiology, such that lifelong maintenance and/or replication of the virus within the infected host is guaranteed. In addition, several of these homologs were reported to have a major impact in the pathogenesis of infection. In this review, the current state of knowledge on the CMV vCKs and vGPCRs will be discussed.

Viral chemokine receptors and chemokines in human cytomegalovirus trafficking and interaction with the immune system. CMV chemokine receptors.

The ubiquitous, opportunistic pathogen human cytomegalovirus (CMV) encodes several proteins homologous to those of the host organism. Four different CMV genes encode chemokine receptor-like peptides. These genes, UL33, UL78, US27, and US28, are expressed at various stages of infection in vitro. Their functions remain largely unknown. To date, chemokine binding and signalling has only been demonstrated for the US28 gene product. Putative ligands for the other CMV-encoded chemokine receptors are discussed on basis of phylogenetic analysis. The potential roles of these receptors in virus trafficking, persistence, and immune evasion are summarized. Similarly, modulation of expression of the host chemokines IL-8, MCP-1a and RANTES in relation to viral dissemination and persistence is reviewed.

Human cytomegalovirus chemokine receptor gene US28 is transcribed in latently infected THP-1 monocytes.

The human cytomegalovirus (HCMV) US28 gene product, pUS28, is a G protein-coupled receptor that interacts with both CC and CX(3)C chemokines. To date, the role of pUS28 in immune evasion and cell migration has been studied only in cell types that can establish productive HCMV infection. We show that HCMV can latently infect THP-1 monocytes and that during latency US28 is transcribed. We also show that the transcription is sustained during differentiation of the THP-1 monocytes. Since cells expressing pUS28 were previously shown to adhere to immobilized CX(3)C chemokines (C. A. Haskell, M. D. Cleary, and I. F. Charo, J. Biol. Chem. 275:34183-34189, 2000), we hypothesize that latently infected circulating monocytes express pUS28, thereby enabling adhesion of these cells to CX(3)C-exposing endothelium. Consequently, the US28-encoded chemokine receptor may play an important role in dissemination of latent HCMV.

The r144 major histocompatibility complex class I-like gene of rat cytomegalovirus is dispensable for both acute and long-term infection in the immunocompromised host.

The rat cytomegalovirus (RCMV) r144 gene encodes a polypeptide homologous to major histocompatibility complex class I heavy chains. To study the role of r144 in virus replication, an RCMV r144 null mutant strain (RCMVDeltar144) was generated. This strain replicated with efficiency similar to that of wild-type (WT) RCMV in vitro. Additionally, WT RCMV and RCMVDeltar144 were found not to differ in their replication characteristics in vivo. First, the survival rate was similar among groups of immunosuppressed rats infected with either RCMVDeltar144 or WT RCMV. Second, the dissemination of virus did not differ in either RCMVDeltar144- or WT RCMV-infected, immunosuppressed rats, either in the acute phase of infection or approximately 1 year after infection. These data indicate that the RCMV r144 gene is essential neither for virus replication in the acute phase of infection nor for long-term infection in immunocompromised rats. Interestingly, in a local infection model in which footpads of immunosuppressed rats were inoculated with virus, a significantly higher number of infiltrating macrophage cells as well as of CD8(+) T cells was observed in WT RCMV-infected paws than in RCMVDeltar144-infected paws. This suggests that r144 might function in the interaction with these leukocytes in vivo.

Deletion of the R78 G protein-coupled receptor gene from rat cytomegalovirus results in an attenuated, syncytium-inducing mutant strain.

The rat cytomegalovirus (RCMV) R78 gene belongs to an uncharacterized class of viral G protein-coupled receptor (GCR) genes. The predicted amino acid sequence of the R78 open reading frame (ORF) shows 25 and 20% similarity with the gene products of murine cytomegalovirus M78 and human cytomegalovirus UL78, respectively. The R78 gene is transcribed throughout the early and late phases of infection in rat embryo fibroblasts (REF) in vitro. Transcription of R78 was found to result in three different mRNAs: (i) a 1.8-kb mRNA containing the R78 sequence, (ii) a 3.7-kb mRNA containing both R77 and R78 sequences, and (iii) a 5.7-kb mRNA containing at least ORF R77 and ORF R78 sequences. To investigate the function of the R78 gene, we generated two different recombinant virus strains: an RCMV R78 null mutant (RCMVDeltaR78a) and an RCMV mutant encoding a GCR from which the putative intracellular C terminus has been deleted (RCMVDeltaR78c). These recombinant viruses replicated with a 10- to 100-fold-lower efficiency than wild-type (wt) virus in vitro. Interestingly, unlike wt virus-infected REF, REF infected with the recombinants develop a syncytium-like appearance. A striking difference between wt and recombinant viruses was also seen in vivo: a considerably higher survival was seen among recombinant virus-infected rats than among RCMV-infected rats. We conclude that the RCMV R78 gene encodes a novel GCR-like polypeptide that plays an important role in both RCMV replication in vitro and the pathogenesis of viral infection in vivo.

Molecular mimicry by cytomegaloviruses. Function of cytomegalovirus-encoded homologues of G protein-coupled receptors, MHC class I heavy chains and chemokines.

Cytomegaloviruses (CMVs) are well known for their high prevalence rate within host populations as well as their ability to induce lifelong infections. To maintain a persistent and stable relationship with their host, CMVs have evolved various molecular mechanisms to both control host cell metabolism and evade immune surveillance. Among the viral gene products that are likely to be involved in these processes are homologues of cellular G protein-coupled receptors, MHC class I molecules and chemokines. The viral genes encoding these homologues have probably been pirated by the viruses during a long pathogen/host coevolution. In this report, we will discuss the possible functions of these homologues in the pathogenesis of CMV infections.

The Maastricht strain and England strain of rat cytomegalovirus represent different betaherpesvirus species rather than strains.

The major immediate early (MIE) locus of the Maastricht strain of rat cytomegalovirus (RCMV) was found to comprise five exons of which the first is noncoding. The first three exons are spliced to either exon 4, generating IE1, or exon 5, generating IE2. An additional splicing event unique to RCMV (Maastricht) was identified in exon 5, resulting in a 466-bp deletion. IE1 transcripts were detected exclusively during the IE phase of infection in vitro, whereas IE2 transcripts were detected during both the IE and late phase of infection. The similarities between amino acid sequences derived from the MIE gene of RCMV (Maastricht) and murine cytomegalovirus are low (22 and 37% for IE1 and IE2, respectively). Surprisingly, the similarities between the MIE proteins of RCMV (Maastricht) and the England strain of RCMV are also low (23 and 32% for IE1 and IE2, respectively). This suggests that these RCMV strains represent different betaherpesvirus species rather than strains. This is underscored by the difference between both viruses in genome size as well as growth characteristics. The existence of two different RCMV-like species might have important implications for the use of these viruses as models for human cytomegalovirus.

The R33 G protein-coupled receptor gene of rat cytomegalovirus plays an essential role in the pathogenesis of viral infection.

We have identified a rat cytomegalovirus (RCMV) gene that encodes a G-protein-coupled receptor (GCR) homolog. This gene (R33) belongs to a family that includes the human cytomegalovirus UL33 gene. R33 was found to be transcribed during the late phase of RCMV infection in rat embryo fibroblasts. Unlike the mRNAs from all the other members of the UL33 family that have been studied to date, the R33 mRNA is not spliced. To study the function of the R33 gene, we constructed an RCMV strain in which the R33 open reading frame is disrupted. The mutant strain (RCMV deltaR33) did not show differences in replication from wild-type RCMV upon infection of several rat cell types in vitro. However, marked differences were seen between the mutant and wild-type strain in the pathogenesis of infection in immunocompromised rats. First, the mutant strain induced a significantly lower mortality than the wild-type virus did. Second, in contrast to wild-type RCMV, the mutant strain did not efficiently replicate in the salivary gland epithelial cells of immunocompromised rats. Although viral DNA was detected in salivary glands of RCMV deltaR33-infected rats up to 14 days postinfection, it could not be detected at later time points. This indicates that although the strain with R33 deleted is probably transported to the salivary glands in a similar fashion to that for wild-type virus, the mutant virus is not able to either enter or replicate in salivary gland epithelial cells. We conclude that the RCMV R33 gene plays a vital role in the pathogenesis of infection.