Showing papers by "Stefan Monecke published in 2018"

Antimicrobial resistance in Enterobacteriaceae from healthy broilers in Egypt: emergence of colistin-resistant and extended-spectrum β-lactamase-producing Escherichia coli .

Abstract: Poultry remains one of the most important reservoir for zoonotic multidrug resistant pathogens. The global rise of antimicrobial resistance in Gram-negative bacteria is of reasonable concern and demands intensified surveillance. In 2016, 576 cloacal swabs were collected from 48 broiler farms located in five governorates in northern Egypt. Isolates of Enterobacteriaceae could be cultivated on different media and were identified by MALDI-TOF MS and PCR. Escherichia coli isolates were genotyped by DNA-microarray-based assays. The antimicrobial susceptibility to 14 antibiotics was determined and resistance-associated genes were detected. The VITEK-2 system was applied for phenotypical confirmation of extended-spectrum β-lactamase-producing isolates. The determination of colistin resistance was carried out phenotypically using E-test and genotypically using PCR for detection of the mcr-1 gene. Out of 576 samples, 72 representatives of Enterobacteriaceae were isolated and identified as 63 E. coli (87.5%), 5 Enterobacter cloacae (6.9%), 2 Klebsiella pneumoniae (2.8%) and 2 Citrobacter spp. (2.8%). Seven out of 56 cultivated E. coli (12.5%) were confirmed as ESBL-producing E. coli and one isolate (1.8%) as ESBL/carbapenemase-producing E. coli. Five out of 63 E. coli isolates (7.9%) recovered from different poultry flocks were phenotypically resistant to colistin and harboured mcr-1 gene. This is the first study reporting colistin resistance and emergence of multidrug resistance in Enterobacteriaceae isolated from healthy broilers in the Nile Delta region, Egypt. Colistin-resistant E. coli in poultry is of public health significance. The global rise of ESBL- and carbapenemase-producing Gram-negative bacteria demands intensified surveillance. ESBL-producing E. coli in poultry farms in Egypt are of major concern that emphasizes the possibility of spread of such strains to humans. The results also reinforce the need to develop strategies and to implement specific control procedures to reduce the use of antibiotics.

Emerging variants of methicillin-resistant Staphylococcus aureus genotypes in Kuwait hospitals.

Abstract: Background Frequent changes in the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) occurring worldwide demand regular surveillance to study their composition and distribution in healthcare facilities. We investigated the genotypic characteristics of MRSA obtained in Kuwait hospitals to better understand their clonal distribution. Materials and methods A total of 1,327 MRSA isolates obtained from clinical samples in 13 Kuwait hospitals from 1 January to 31 December 2016 were investigated using antibiogram, SCCmec typing, spa typing and DNA microarray. Results The isolates belonged to six SCCmec types with the majority belonging to type IV (658; 49.5%) and type V (355; 26.7%). Two hundred and sixty-one spa types were identified with spa types t688, t304, t860, t127, t044, t311, t002, t223, t267, t019, t3841, t005, t084, t852, and t657 constituting 51.0% (n = 677) of the isolates. Among the 1,327 MRSA isolates, 102 (7.68%) isolates were identified as novel variants of internationally recognized MRSA clones. These 102 isolates were investigated further and belonged to 14 clonal complexes (CCs) with CC361 (32; 32.3%), CC30 (15; 14.7%), CC22 (13; 12.7%) and CC1 (11, 10.7%) as the dominant CCs. Eighty-one (79.4%) of the novel isolates harbored SCCmec IV or V+fusC composite genetic elements. Four isolates (3.9%) harbored unusual combinations of ccr and mec complexes comprising of CC6-MRSA [IV+fusC+ccrC], CC97-MRSA [V/VT+fusC+ccrAB2], CC121-MRSA [V/VT+fusC+ccrB4] and CC1-MRSA-pseudoSCCmec [class B mec+fusc+ccrAB1]. Forty-six (45.1%) of these isolates were positive for PVL and 89 (87.2%) were resistant to fusidic acid mediated by fusC. Conclusions The study showed the emergence of novel variants of previously recognized MRSA genotypes with unusual genetic characteristics including high prevalence of PVL and fusidic acid resistance in Kuwait hospitals. This has added to the dynamic lists of known variations in MRSA genomes which can impose serious challenges for infection control and treatment of MRSA infections.

Molecular Typing of ST239-MRSA-III From Diverse Geographic Locations and the Evolution of the SCCmec III Element During Its Intercontinental Spread.

Abstract: ST239-MRSA-III is probably the oldest truly pandemic MRSA strain, circulating in many countries since the 1970s. It is still frequently isolated in some parts of the world although it has been replaced by other MRSA strains in, e.g., most of Europe. Previous genotyping work (Harris et al., 2010; Castillo-Ramirez et al., 2012) suggested a split in geographically defined clades. In the present study, a collection of 184 ST239-MRSA-III isolates, mainly from countries not covered by the previous studies were characterized using two DNA microarrays (i) targeting an extensive range of typing markers, virulence and resistance genes and (ii) a SCCmec subtyping array. Thirty additional isolates underwent whole-genome sequencing (WGS) and, together with published WGS data for 215 ST239-MRSA-III isolates, were analyzed using in-silico analysis for comparison with the microarray data and with special regard to variation within SCCmec elements. This permitted the assignment of isolates and sequences to 39 different SCCmec III subtypes, and to three major and several minor clades. One clade, characterized by the integration of a transposon into nsaB and by the loss of fnbB and splE was detected among isolates from Turkey, Romania and other Eastern European countries, Russia, Pakistan, and (mainly Northern) China. Another clade, harboring sasX/sesI is widespread in South-East Asia including China/Hong Kong, and surprisingly also in Trinidad & Tobago. A third, related, but sasX/sesI-negative clade occurs not only in Latin America but also in Russia and in the Middle East from where it apparently originated and from where it also was transferred to Ireland. Minor clades exist or existed in Western Europe and Greece, in Portugal, in Australia and New Zealand as well as in the Middle East. Isolates from countries where this strain is not epidemic (such as Germany) frequently are associated with foreign travel and/or hospitalization abroad. The wide dissemination of this strain and the fact that it was able to cause a hospital-borne pandemic that lasted nearly 50 years emphasizes the need for stringent infection prevention and control and admission screening.

Intra-Hospital, Inter-Hospital and Intercontinental Spread of ST78 MRSA From Two Neonatal Intensive Care Unit Outbreaks Established Using Whole-Genome Sequencing.

Abstract: From 2009 to 2011 [transmission period (TP) 1] and 2014 to 2017 (TP2), two outbreaks involving community-associated clonal complex (CC) 88-MRSA spa types t186 and t786, respectively, occurred in the Neonatal Intensive Care Unit (NICU) of an Irish hospital (H1). This study investigated the relatedness of these isolates, their relationship to other CC88 MRSA from Ireland and their likely geographic origin, using whole-genome sequencing (WGS). All 28 CC88-MRSA isolates identified at the Irish National MRSA Reference Laboratory between 2009 and 2017 were investigated including 20 H1 patient isolates, two H1 isolates recovered from a single healthcare worker (HCW) 2 years apart, three patient isolates from a second hospital (H2) and one patient isolate from each of three different hospitals (H3, H4, and H5). All isolates underwent DNA microarray profiling. Thirteen international isolates with similar microarray profiles to at least one Irish isolate were selected from an extensive global database. All isolates underwent Illumina MiSeq WGS. The majority of Irish isolates (25/28; all H1 isolates, two H2 isolates and the H3 isolate) were identified as ST78-MRSA-IVa and formed a large cluster, exhibiting 1-71 pairwise allelic differences, in a whole-genome MLST-based minimum spanning tree (MST) involving all Irish isolates. A H1/H2, H1/H3, and H1 HCW/patient isolate pair each exhibited one allelic difference. The TP2 isolates were characterised by a different spa type and the loss of hsdS. The three remaining Irish isolates (from H2, H4, and H5) were identified as ST88-MRSA-IVa and dispersed at the opposite end of the MST, exhibiting 81-211 pairwise allelic differences. Core-genome MLST and sequence-based plasmid analysis revealed the recent shared ancestry of Irish and Australian ST78-MRSA-IVa, and of Irish and French/Egyptian ST88-MRSA-IVa. This study revealed the homogeneity of isolates recovered during two NICU outbreaks (despite spa type and hsdS carriage variances), HCW involvement in the outbreak transmission chain and the strain's spread to two other Irish hospitals. The outbreak strain, CC88/ST78-MRSA-IVa, was likely imported from Australia, where it is prevalent. CC88/ST88-MRSA-IVa was also identified in Irish hospitals and was likely imported from Africa, where it is predominant, and/or a country with a large population of African descent.

Prevalence of carbapenemase-producing organisms at the Kidney Center of Rawalpindi (Pakistan) and evaluation of an advanced molecular microarray-based carbapenemase assay

Abstract: Aim A DNA microarray-based assay for the detection of antimicrobial resistance (AMR) genes was used to study carbapenemase-producing organisms at the Kidney Center of Rawalpindi, Pakistan. Methods The evaluation of this assay was performed using 97 reference strains with confirmed AMR genes. Testing of 7857 clinical samples identified 425 Gram-negative bacteria out of which 82 appeared carbapenem resistant. These isolates were analyzed using VITEK-2 for phenotyping and the described AMR assay for genotyping. Results The most prevalent carbapenemase gene was blaNDM and in 12 isolates we detected two carbapenemase genes (e.g., blaNDM/blaOXA-48). Conclusion Our prevalence data from Pakistan show that - as in other parts of the world - carbapenemase-producing organisms with different underlying resistance mechanisms are emerging, and this warrants intensified and constant surveillance.

Hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) from Pakistan: molecular characterisation by microarray technology

Abstract: The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in Pakistan is known to be high, but very few studies have described the molecular epidemiology of the different MRSA clones circulating in the country. Forty-four MRSA isolates were collected from two tertiary care hospitals of the Rawalpindi district of Pakistan. All strains were identified by a conventional phenotypic method and then subjected to genotyping by microarray hybridisation. Six clonal complexes (CCs) and 19 strains were identified. The most commonly identified strains were: (i) Panton–Valentine leucocidin (PVL)-positive CC772-MRSA-V, “Bengal Bay Clone” (ten isolates; 22.3%), (ii) ST239-MRSA [III + ccrC] (five isolates) and (iii) a CC8-MRSA-IV strain, as well as CC6-MRSA-IV (both with four isolates; 9.1% each). Several of the strains detected indicated epidemiological links to the Middle Eastern/Arabian Gulf region. Further studies are needed to type MRSA from countries with less known epidemiology and to monitor the distribution and spread of strains, as well as possible links to global travel, migration and commerce.

Shiga Toxin-Producing Escherichia coli Infection in Jönköping County, Sweden: Occurrence and Molecular Characteristics in Correlation With Clinical Symptoms and Duration of stx Shedding.

Abstract: Shiga toxin-producing Escherichia coli (STEC) cause bloody diarrhea (BD), hemorrhagic colitis (HC), and even hemolytic uremic syndrome (HUS). In Nordic countries, STEC are widely spread and usually associated with gastrointestinal symptoms and HUS. The objective of this study was to investigate the occurrence of STEC in Swedish patients over 10 years of age from 2003 through 2015, and to analyze the correlation of critical STEC virulence factors with clinical symptoms and duration of stx shedding. Diarrheal stool samples were screened for presence of stx by real-time PCR. All STEC isolates were characterized by DNA microarray assay and PCR to determine serogenotypes, stx subtypes, and presence of intimin gene eae and enterohaemolysin gene ehxA. Multilocus sequencing typing (MLST) was used to assess phylogenetic relationships. Clinical features were collected and analyzed using data from the routine infection control measures in the county. A total of 14,550 samples were enrolled in this 12-years period study, and 175 (1.2%) stools were stx positive by real-time PCR. The overall incidence of STEC infection was 4.9 cases per 100,000 person-years during the project period. Seventy-five isolates, with one isolate per sample were recovered, among which 43 were from non-bloody stools, 32 from BD, and 3 out of the 75 STEC positive patients developed HUS. The presence of stx2 in both stools and isolates were associated with BD (p = 0.008, p = 0.05), and the presence of eae in isolates was related to BD (p = 0.008). The predominant serogenotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Isolates from HUS were O104:H4 and O98: H21 serotypes. Phylogenetic analysis revealed our strains were highly diverse, and showed close relatedness to HUS-associated STEC collection strains. In conclusion, the presence of stx2 in stool was related to BD already at the initial diagnostic procedure, thus could be used as risk predictor at an early stage. STEC isolates with stx2 and eae were significantly associated with BD. The predominant serotypes associated with BD were O157:H7, O26:H11, O121:H19, and O103:H2. Nevertheless, the pathogenic potential of other serotypes and genotypes should not be neglected.

Molecular Analysis of Two Different MRSA Clones ST188 and ST3268 From Primates (Macaca spp.) in a United States Primate Center.

Abstract: Methicillin-resistant Staphylococcus aureus (MRSA) were identified in macaques, their environmental facility, and nasal cultures of personnel from the Washington National Primate Research Center [WaNPRC] and included MRSA ST188 SCCmec IV and MRSA ST3268 SCCmec V. The aim of the current study was to determine the carriage of virulence genes, antibiotic resistance genes, and other characteristics of the primate MRSA isolates to determine if there were any obvious differences that would account for differences in transmission within the WaNPRC facility. In total, 1,199 samples from primates were tested for the presence of MRSA resulting in 158 MRSA-positive samples. Fifteen ST188 isolates (all from Macaca nemestrina) and nine ST3268 (four from Macaca mulatta, two from Macaca fascicularis, three from M. nemestrina), were selected for further characterization. All but one of the 15 ST188 isolates had spa type t189 and the remaining one had spa type t3887. These isolates were resistant to β-lactams [blaZ, mecA], macrolides/lincosamides [erm(B)], aminoglycosides [aacA-aphD], and fluoroquinolones. Five isolates were additionally resistant to tetracyclines [tet(K)] and had elevated MICs for benzalkonium chloride [qacC]. In comparison, the nine ST3268 isolates had the related spa types t15469 (n = 5) and t13638 (n = 4). All nine ST3268 isolates were resistant to β-lactams [blaZ, mecA], and tetracyclines [tet(K)]. Some isolates were additionally resistant to aminoglycosides [aacA-aphD], fluoroquinolones and/or showed elevated MICs for benzalkonium chloride [qacC]. In contrast to the ST188 isolates, the ST3268 isolates had the enterotoxin gene cluster egc [seg, sei, selm, seln, selo, selu] and enterotoxin genes sec and sel. The two clones have differences regarding their spa types, virulence and antibiotic resistance genes as well as ST and SCCmec types. However, the data presented does not provide insight into why ST188 spreads easily while ST3268 did not spread within the WaNPRC in-house primates.

Genotyping of Staphylococcus aureus associated with nasal colonization among healthcare workers using DNA microarray.

Abstract: INTRODUCTION Healthcare workers (HCWs) colonized with Staphylococcus aureus may serve as a reservoir of infection. This study was carried to determine the genetic make-up of S. aureus nasal colonizers in HCWs. METHODOLOGY Nasal swabs were obtained from 93 HCWs and molecular characterization of identified S. aureus isolates was carried out using the StaphyType DNA microarray (Alere Technologies GmbH, Jena, Germany). RESULTS Twenty-nine HCWs (31%) were colonized with S. aureus (MSSA = 23; MRSA = 6). Thus the overall MRSA carriage rate was 6.5% (n/N = 6/93) and 20.7% (n/N = 6/29) of those colonized with S. aureus harboured MRSA. The S. aureus isolates belonged to 16 clonal complexes (CC). MSSA isolates included three each for CC15, CC188, ST2867; two each for CC5, CC97, CC367 as well as one each for CC1, CC8, CC30, CC45, CC101, CC121, ST291/813 and CC1153. The staphylococcal cassette chromosome recombinase genes ccrA-1; ccrB-1 and the fusidic acid resistance gene (fusC) were present in two MSSA isolates (CC1 and CC8). The six MRSA isolates included CC5-MRSA-[VI+fusC] (n = 2); one each of CC5-MRSA-V; CC22-MRSA-IV (tst1+); CC80-MRSA-IV [pvl+] ("European CA-MRSA Clone") and CC97-MRSA-[V+fusC]. CONCLUSION There is wide clonal diversity of S. aureus colonizers with associated high MRSA carriage among the HCWs. The presence of genetically stable MSSA isolates with the capability to transform into MRSA isolates is of concern.

Emergence and global spread of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

Abstract: The global spread of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here we demonstrate the recent origin and global spread from the Indian subcontinent of a multidrug resistant Staphylococcus aureus lineage, sequence type 772 (Bengal Bay clone). Short-term outbreaks occurred following intercontinental transmission, typically associated with travel and family contacts, but ongoing endemic transmission was uncommon. Instrumental in the emergence of a single dominant clade in the early 1990s was the acquisition of a multidrug resistance integrated plasmid that did not appear to incur a significant fitness cost. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological and virulence potential of community-associated clones.

Rethinking the Molecular Diagnostics for Methicillin-Resistant Staphylococcus aureus.

Abstract: To the Editor—Infections due to methicillin-resistant Staphylococcus aureus (MRSA) strains are a common clinical problem that causes a high burden of disease and that often requires long-term treatment. MRSA rates vary in different countries, with rates being high in the United States, Middle and Far Eastern countries, and Southern Europe but relatively low in, for example, Northern Europe. Encoded by different alleles of the mecA gene, an alternate penicillin-binding protein (PBP2a) is expressed in MRSA conferring resistance to β-lactam antibiotics (except ceftaroline and ceftobiprole). mecA is situated on a mobile genetic element named staphylococcal cassette chromosome mec (SCCmec). Currently, this mobile element occurs as 12 different types in S. aureus as well as in other clinically relevant staphylococci such as S. haemolyticus and S. epidermidis. In 2011, a divergent mecA homologue was discovered and subsequently designated mecC. mecC MRSA has been isolated from patients from Western and Central Europe. It has also been detected in different domestic and wild animals including cattle, sheep, hedgehogs, storks, and fox. The reservoir of mecC strains is still unknown, although zoonotic transmission from cows or sheep is believed to play a major role. The mecC gene was detected in various rare lineages of S. aureus in multilocus sequence type (MLST) clonal complexes (CC) 49, 130, 425, 599, and 1943. The amino acid identity between mecCand mecA-encoded PBP2a is only 63%. While themecC gene itself does not mediate resistance to penicillin, it is frequently accompanied by the β-lactamase blaZ, which is part of the SCCmec XI element that does confer resistance to penicillin. Previous studies found that mecC also mediates resistance to oxacillin and cefoxitin. We report a 59-year-old male patient with a postthrombotic ulcer on his right lower leg measuring 3 × 3 cm. This patient had no other concomitant disease and no prior hospital admission. The bacterial culture of the wound swab was analysed with Vitek 2 (bioMérieux, Nürtingen, Germany) and broth microdilution according to EUCAST break points. The isolate was further analyzed using the S. aureus Genotyping Kit 2.0 (Alere Technologies, Jena, Germany). After repetitive wound debridement, bacterial culture of a wound swab revealed an S. aureus isolate resistant to benzylpenicillin, oxacillin, and cefoxitin in the routine susceptibility test. Other tested antibiotics typically used for treatment of S. aureus (eg, glycopeptides and chinolones) were found to be susceptible. The MRSA isolate carried the mecC gene was negative for Panton-Valentine leukocidin (PVL) and was assigned to the CC130 lineage. The isolate belonged to agr group III and capsule type 8, and themecC gene was present as a part of the SCCmecXI element together with the distinct blaZ. Despite the association of the mecC gene to zoonotic transmission, the patient reported no contact with livestock or any other potential animal source. The patient was admitted to the hospital for treatment of the leg ulcer and was simultaneously MRSA sanitized according to the German guidelines on 5 consecutive days with an octenidin-containing wound compresses for 20 minutes per day, mupirocin nasal ointment, throat flushing with chlorhexidine, and skin washing with octenidin. After waiting for 3 days, control swabs were taken on 3 consecutive days from the nose, throat, skin, and wound. None of these 12 swabs were positive for S. aureus, which was interpreted as evidence for successful sanitization exclusively with topical treatment. During the following 2 years, all 10 swabs taken for diagnostic and screening purposes remained negative for S. aureus. The prevalence ofmecC in humans and animals is still low: the value reported in a meta-analysis was 0.009% (95% confidence interval = 0.055–0.013%) of MRSA, which corresponds to an order of magnitude range of 1:100 to 1:1000 of typed MRSA. While mecC strains in humans do not appear to be particularly virulent, cohort studies are missing and case reports are sparse. Currently, there is no recommendation on treatment of mecC MRSA. With regard to treatment options, minimum inhibitory concentrations (MICs) for methicillin and cefoxitin are usually lower than in conventional MRSA, but these compounds might not be effective. Penicillin is an inhibitor for themecC gene product, but becausemecC is accompanied by a specific penicillinase (also carried on the SCCmec XI element), monotherapy is not a treatment option. However, in vitro and in vivo models have shown an effective combination with a β-lactamase inhibitor. The patient was treated as an mecA MRSA case to overcome the risk of treatment failures in case of susceptible or intermediate oxacillin MICs. The current setup to diagnose mecA/mecC of MRSA is multifaceted and time-consuming. Because of its divergent sequence, mecC and its gene product cannot be detected by all assays designed to identifymecA/PBP2a, and it has been suggested that mecC is largely underreported. The discrepancy between phenotypic resistance and mecA-negative molecular or proteinbased confirmatory tests might delay reporting to the physician and, thus, also delay the administration of an appropriate treatment. Hence, the discovery of mecC has resulted in the recognition of the need to redesign diagnostic tests. In summary, clinicians and microbiologists should be aware of the changing facets of MRSA infections, particularly the emergence of mecC MRSA-conferred resistance against oxacillin. MRSA resistance warrants further investigation, especially in cases of discrepant testing results. Because the clinical experience is limited to case reports (probably due to the underreporting ofmecCMRSA), amecCMRSA infection should be treated as anmecAMRSA infection to avoid treatment failure.