Occurrence of class 1 integrons in uropathogenic fluoroquinolone‐resistant clinical Escherichia coli isolates from Jamaica

Quinolone resistance is generally caused by chromosomal mutations, but has been more recently found associated with the plasmid‐mediated qnr genes. The objective of this study was to screen and analyse polymorphisms of integrons in clinical isolates of Escherichia coli in Jamaica. Previous studies in Jamaica identified fluoroquinolone resistance in predominantly uropathogenic E. coli clinical isolates: 45% harbouring qnrA, qnrB and/or qnrS, and 17% were (Extended‐spectrum beta‐lactamase) ESBL‐producers. These isolates were analysed for the presence and variation of class 1 and 2 integrase genes, 5′‐ and 3′‐ conserved segments and the Orf513 recombinase gene by primer‐specific polymerase chain reaction (PCR) and restriction fragment‐length polymorphism (RFLP). Results indicated integron‐encoded integrases in 93% of isolates primarily harbouring class 1 integrase genes; four of 58 isolates carried both classes. The Orf513 and 5′‐ and 3′‐conserved segment (CS) regions were identified in 83% and 55% of the isolates respectively. RFLP evaluation of the 5′‐ and 3′‐CS regions in int1‐positive strains yielded two main types. The reduced diversity, but wide dispersion of class 1 integrons harbouring qnr genes may give rise to the conservation of the mobile genetic elements in which they are carried.     

Ability of the GENSPEED® MRSA test kit to detect the novel mecA homologue mecC in Staphylococcus aureus

The aim of this study was to evaluate the GENSPEED^® MRSA Test Kit that in addition to the traditional MRSA gene mecA, also incorporates a probe for detection of the newly described mecA homologue mecC. So far only one commercial system is able to detect this new gene. The specific objective was to evaluate the ability of the kit to detect and separate Stapyhylococcus aureus containing either mecA, mecC or no methicillin‐resistance determinant. Ninety‐five MRSA isolates from humans were included in the test and additional mecC‐positive isolates from animals and methicillin‐sensitive S. aureus were tested. The kit demonstrated 100% sensitivity and 100% specificity compared to a standard PCR method. The kit provides the ability to perform rapid and reliable detection of both mecA‐MRSA and mecC‐MRSA.     

Biofilm formation of ica operon‐positive Staphylococcus epidermidis from different sources

Information on the prevalence of biofilm‐related factors (PIA, Bhp, Aap, Embp) in Staphylococcus epidermidis of animal origin is scarce. In this study, 263 S. epidermidis isolates of diverse origin (animal, farmers, patients, and laboratory staff) were investigated for the presence of the ica operon (icaRADBC). The icaRADBC‐positive isolates were further characterized by means of biofilm formation, presence of other biofilm‐related genes, antimicrobial resistance, and population structure. Of all isolates, 28.5% (n = 75) were icaRADBC‐positive, including 16.5% of animal origin, 29.1% farmer isolates, and 44.6% hospital‐associated isolates (including patients and laboratory staff isolates). Most icaRADBC‐positive isolates carried embp (n = 73), aap (n = 57), bhp (n = 22), and IS256 (n = 29). Statistical differences were found between animal and patient isolates for the presence of icaRADBC, bhp, and aap. No statistically significant relation was found between the presence of one or more genes and the level of biofilm formation. Most icaRADBC‐positive isolates belonged to the clonal complex 5 (formerly 2) and most sequence types corresponded to types previously observed in community and nosocomial S. epidermidis populations. Although the prevalence of S. epidermidis in the nasal cavity of bovines and poultry is low, some isolates belong to STs related to ica‐positive clinical strains.     

Distribution of Ebp pili among clinical and fecal isolates of Enterococcus faecalis and evaluation for human platelet activation

Although Enterococcus faecalis is known as normal flora in colon, it is also amongst the most common causative agents of infective endocarditis (IE). Platelet activation resulting from adherence to platelets is an essential step in the pathogenesis of IE. One of the factors proposed in adhesion is endocarditis‐ and biofilm‐ associated pili encoded by ebp operon. The aim of this study was to investigate ebp in isolates from different origins and analyze the potential of isolates to activate human platelets of different donors. The ebp distribution was investigated in E. faecalis from different origin infections (n = 103) and fecal flora (n = 20). Then, selected isolates from blood (n = 5), urine (n = 2), and fecal flora (n = 3) were analyzed by flow cytometry assay for the ability to activate platelets of four different donors. No statistically significant difference was found for the ebp presence between infective and fecal isolates. Also, it was found that the ability for platelet activation is independent of the bacterial origin. However, significant difference was found in platelet activation between different donors. The results suggest that the presence or absence of ebp is not a critical factor for platelet activation by E. faecalis isolates. However, host factors seem to contribute in this activity.     

Molecular characterization and antibiotic resistance of clinical isolates of methicillin‐resistant Staphylococcus aureus obtained from Southeast of Iran (Kerman)

Staphylococcus aureus infections, particularly infections caused by methicillin‐resistant S. aureus (MRSA) strains, are emerging as a major public health problem. The aim of this study was to determine the prevalence of MRSA, antibiotic resistance profile and staphylococcal cassette chromosome mec (SCCmec) type of MRSA isolates obtained from clinical samples. Totally, 162 S. aureus isolates were obtained from clinical samples at three university hospitals in Kerman, Iran from March 2011 to February 2012. All isolates were identified as S. aureus by phenotypic methods and confirmed by PCR amplification of the nuc gene . MRSA isolates were screened by phenotypic tests and confirmed by presence of mecA gene. The minimum inhibitory concentrations (MICs) of the MRSA isolates against antibacterial agents were determined by E‐test. All isolates were analyzed by PCR for the presence of mecA and pvl genes. SCCmec typing of MRSA isolates was performed by multiplex PCR assay. Strain typing was carried out with REP‐PCR. Using mecA gene PCR and phenotypic methods, 56.8% of the isolates were identified as MRSA. All MRSA isolates were susceptible to vancomycin and linezolid. The sensitivity of MRSA isolates to trimethoprim‐sulfamethoxazole, clindamycin, ciprofloxacin, gentamicin, and erythromycin was 70.66, 66.53, 42.4, 38.05, and 29.35%, respectively. The most frequent SCCmec types were type III (48.31%) followed by type V (19.1%), type I (16.85%), and type IV (3.37%). The pvl gene was detected in 3.08% of isolates (two MRSA and three MSSA isolates). REP‐PCR typing divided the 92 MRSA isolates into 10 distinct clusters. Our results indicate that vancomycin and linezolid are the most effective antibacterial agents against MRSA isolates and SCCmec type III is predominant in MRSA strains in this area.     

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh,Iran

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed‐field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER‐1 extended‐spectrum β‐lactamase‐producing P. aeruginosa.     

The susceptibility of Campylobacter concisus to the bactericidal effects of normal human serum

Campylobacter concisus is an emerging pathogen of the gastrointestinal tract that has been associated with Barrett's oesophagus, enteritis and inflammatory bowel disease. Despite having invasive potential in intestinal epithelial cells in‐vitro, bacteraemic cases with C. concisus are extremely scarce, having only been reported once. Therefore, we conducted a serum resistance assay to investigate the bactericidal effects of human complement on C. concisus in comparison to some other Campylobacter species. In total, 22 Campylobacter strains were tested by incubation with normal human serum and subsequent cultivation in microaerobic conditions for 48 hours. Killing time was evaluated by decrease in total CFU over time for incubation with different serum concentrations. Faecal isolates of C. concisus showed inoculum reduction to less than 50% after 30 min. Campylobacter jejuni was sensitive to serum, but killing was delayed and a bacteraemic Campylobacter fetus subsp. fetus isolate was completely serum resistant. Interestingly, sensitivity of enteric C. concisus to human serum was not associated to different faecal‐calprotectin levels. We find that faecal isolates of C. concisus are sensitive to the bactericidal effects of serum, which may explain why C. concisus is not associated to bacteraemia.     

Evaluation of molecular typing methods for identification of outbreak‐associated Neisseria meningitidis isolates

It is essential in an outbreak investigation that strain characterization of Neisseria meningitidis is performed in a rapid and accurate manner. This study evaluated two new molecular typing methods, multiple‐locus variable number tandem repeat analysis (MLVA) and repetitive sequence‐based PCR (rep‐PCR) (DiversiLab; bioMérieux) and compared them with current recommended methodologies. This retrospective study included 36 invasive N. meningitidis serogroup C isolates collected in Sweden 2001 through 2009 and previously subjected to outbreak investigation. All strains were typed with highly variable‐MLVA (HV‐MLVA) and rep‐PCR. The isolates were further characterized by multilocus sequence typing (MLST) and sequencing of the fetA, fHbp, penA, porA and porB genes. The results showed that HV‐MLVA had the highest index of diversity (0.99) and rep‐PCR had the highest congruence (40%) with the currently recommended typing methods. The HV‐MLVA correlated best to the spatiotemporal connections and had the overall highest Adjusted Wallace coefficients, suggesting that HV‐MLVA can predict the results of the other typing methods in the study. We therefore suggest that after initial confirmation of species, serogroup and genosubtype, HV‐MLVA should be used as the most discriminatory method for first hand investigation of N. meningitidis serogroup C isolates.     

Emergence of type I restriction modification system‐negative emm1 type Streptococcus pyogenes clinical isolates in Japan

Streptococcus pyogenes emm1 type is the dominant cause of streptococcal toxic shock syndrome (STSS) in Japan and many other developed countries. Recently, the number of STSS patients in Japan was reported to be increasing. Hence, we analyzed the S. pyogenes clinical isolates detected in Japan after 2005. We found that the regions encoding the Spy1908–1910 two‐component regulatory system and the adjacent type I restriction modification system were deleted in some emm1 type isolates. The isolates with the deletion were detected only in the emm1 strains that were isolated between 2010 and 2013, but not before 2010. Twenty‐six of 46 (56.5%) emm1 type isolates were isolated in 2010–2013, and among these isolates, five of seven (71.4%) emm1 type STSS isolates were shown to have that deletion. PFGE and PCR analysis for the presence of several pyrogenic exotoxin‐related genes suggested that the emm1 isolates with and without the deletion shared the same genetic background. The emm1 isolates with the deletion could incorporate exogenous plasmids by experimental electroporation transformation far more efficiently. These results suggested that the novel emm1 isolates have occupied a fairly large part of total emm1 isolates.     

Phagocytosis and cytokine response to rough and smooth colony variants of Mycobacterium abscessus by human peripheral blood mononuclear cells

Mycobacterium abscessus is a non‐tuberculous mycobacteria able to cause opportunistic infections in selected patient groups. During the last decades it has emerged as a cause of chronic pulmonary infection in patients with cystic fibrosis (CF). M. abscessus strains exhibit either smooth or rough colony morphology. Strains exhibiting the rough phenotype more often cause pulmonary infections in CF patients than did the smooth ones. Here, we examined phagocytosis and production of cytokines by human peripheral blood mononuclear cells, in response to M. abscessus strains with smooth and rough colony phenotype. The rough isolates all formed multicellular cords, similar to what is observed in Mycobacterium tuberculosis. Monocytes were generally unable to internalize these rough cord isolates, in contrast with the smooth ones. Furthermore, the rough M. abscessus strains induced a distinct cytokine profile differing from that induced by the smooth ones. Rough isolates induced significantly less IL‐10 and tumour necrosis factor compared to smooth strains, but more IL‐1β. Both varieties induced equal amounts of IFN‐γ, IL‐17, IL‐23, IL‐6, IL‐8 and equally little IL‐12. The ability to withstand phagocytosis might be a virulence factor contributing to the capacity of rough M. abscessus strains to give persistent pulmonary infections.     

Survival in the environment is a possible key factor for the expansion of Escherichia coli strains producing extended‐spectrum β‐lactamases

Acquired resistance to cephalosporins in Enterobacteriaceae is a global problem. After an outbreak at Uppsala University Hospital of extended‐spectrum β‐lactamase (ESBL)‐positive Klebsiella pneumoniae producing CTX‐M‐15, there was a shift from AmpC to ESBL production among Escherichia coli isolates. To explore the basis for this epidemiological shift, 46 E. coli isolates (ESBLs, n = 23; AmpC, n = 23) were characterized with regard to genetic relatedness, β‐lactamase, replicon and integron types, antibiotic resistance profiles, and genes encoding virulence factors. In addition, the survival in the environment and on hospital‐associated materials was analysed. CTX‐M‐15 was the most frequent ESBL (78%). Only three (13%) of the AmpC enzymes were harboured on plasmids (CMY‐2, DHA‐1). Independent of plasmid‐mediated beta‐lactamase, IncF plasmids predominated and only class I integrons were detected. The ESBL producers carried more virulence genes (p = 0.04), exhibited a broader resistance phenotype (p = 0.01) and survived significantly longer (p = 0.03) on different materials than the AmpC‐producing isolates. In conclusion, ESBL‐producing isolates had properties which are likely to augment their competitiveness. Apart from antibiotic resistance and virulence factors, extended survival in the environment could be a selective trait for successful ESBL‐producing E. coli strains.     

High frequency of multidrug‐resistant Staphylococcus aureus with SCCmec type III and Spa types t037 and t631 isolated from burn patients in southwest of Iran

Methicilin resistance Staphylococcus aureus (MRSA) infections are the major challenges in hospitals, especially in the burn units. The use of molecular typing methods is essential for tracking the spread of S. aureus infection and epidemiological investigations. The aim of this study was to find the profile of the spa types and also the prevalence of each SCCmec type of S. aureus strains in a central burn hospital in southwest of Iran. A total of 81 non‐duplicate S. aureus were isolated from burn patients between April 2011 and February 2012. The susceptibility of the isolates against 13 different antibiotics was tested by disk agar diffusion (DAD) method. MRSA strains were identified by amplification of mecA gene. Multiplex‐polymerase chain reaction (PCR) technique was used to determine the SCCmec types of MRSA strains and all the S. aureus isolates were typed by spa typing method. Detection of mecA gene showed that 70 (86.4%) of the isolates were MRSA. The highest rate of resistance was observed for penicillin (97.5%) and erythromycin (77.8%). None of the isolates were resistant to vancomycin. Sixty‐seven of the 70 MRSA isolates harbored only SCCmec type III and three untypeable isolates. Five different spa types were detected. The most common spa types were t037 (42.5%) and t631 (34.5%) and were only found in MRSA isolates. Only SCCmec type III was found in burn patients which emphasizes the HA‐MRSA origin of these strains. Only five different spa types identified in this study are in accordance with one SCCmec type which indicates that a limited number of bacterial colons are circulated in the burn unit in this hospital.     

Genotyping of Pseudomonas aeruginosa isolates from lung transplant recipients and aquatic environment–detected in‐hospital transmission

Lung infection with Pseudomonas aeruginosa is common in lung transplant recipients and may lead to severe complications. Bacteriological surveillance aims to detect transmission of microbes between hospital environment and patients. We sought to determine whether genotyping of P. aeruginosa isolates could improve identifications of pathways of infection. From 2004 to 2009, we performed genotyping with multiple‐locus variable number of tandem repeats analysis (MLVA) and pulsed‐field gel electrophoresis (PFGE) of P. aeruginosa isolates cultured from lung transplant recipients at Sahlgrenska University Hospital, Gothenburg. During a small outbreak in 2008, cultivation and genotyping of isolates from sink and drains samples from the hospital ward were performed. Pseudomona aeruginosa from 11/18 patients were genotyped to unique strains. The remaining seven patients were carriers of a P. aeruginosa strain of cluster A genotype. Pseudomona aeruginosa was isolated in 4/8 water samples, typed by MLVA also as cluster A genotype and confirmed by PFGE to be similar or identical to the isolates from four transplanted patients. In conclusion, genotyping of isolates revealed a clonal relationship between patient and water isolates, indicating in‐hospital transmission of P. aeruginosa. We suggest genotyping with MLVA for rapid routine surveillance, with the PFGE method used for extended, confirmatory analyses.     

Motility of Campylobacter concisus isolated from saliva,feces, and gut mucosal biopsies

Campylobacter concisus is an emerging pathogen associated with gastrointestinal disorders such as gastroenteritis and inflammatory bowel diseases (IBD), but the species is also found in healthy subjects. The heterogeneous genome of C. concisus increases the likelihood of varying virulence between strains. Flagella motility is a crucial virulence factor for the well‐recognized Campylobacter jejuni; therefore, this study aimed to analyze the motility of C. concisus isolated from saliva, gut biopsies, and feces of patients with IBD, gastroenteritis, and healthy subjects. The motility zones of 63 isolates from 52 patients were measured after microaerobic growth in soft‐agar plates for 72 hours. The motility of C. concisus was significantly lower than that of Campylobacter jejuni and Campylobacter fetus subsp. fetus. The motility of C. concisus varied between isolates (4–22 mm), but there was no statistical significant difference between isolates from IBD patients and healthy subjects (p = 0.14). A tendency of a larger motility zones was observed for IBD gut mucosa isolates, although it did not reach statistical significance (p = 0.13), and no difference was found between oral or fecal isolates between groups. In conclusion, the varying motility of C. concisus could not be related to disease outcome or colonization sites.     

Antimicrobial resistance,prevalence of resistance genes,and molecular characterization in intestinal Bacteroides fragilis group isolates

Since the level of antimicrobial resistance in Bacteroides fragilis has increased, monitoring the antimicrobial susceptibility could be necessary. The objectives of this study were to (i) investigate the prevalence of species, the occurrence of reduced antimicrobial susceptibility (E‐test method), and antibiotic resistance genes in the B. fragilis group and (ii) evaluate the prevalence of enterotoxigenic B. fragilis and the distribution of bft gene subtypes in hospitalized patients. As many as 475 isolates out of 250 stool samples were detected to be B. fragilis group by using conventional biochemical tests (API‐32A system) and multiplex‐PCR. In addition, 48.2%, 13.9%, 76.6%, and 1.2% of B. fragilis group isolates were resistant (according to EUCAST breakpoint) to piperacillin‐tazobactam, meropenem, clindamycin, and metronidazole, respectively. Six metronidazole‐resistant strains were isolated; B. fragilis (n: 3), B. thetaiotaomicron, B. vulgates, and B. ovatus. The presence of the cfiA, cepA, ermF, and nim genes was observed in 3.8%, 15.9%, 34.1%, and 0.7% of the B. fragilis isolates, respectively. One hundred thirty‐two B. fragilis isolates (27.8%)and 21 B  fragilis isolates (15.9%) turned out to be bft gene positive by multiplex‐PCR; eleven isolates (52.4%) harbored bft‐1, eight isolates (38%) harbored bft‐2 isotypes, and two isolates (9.5%) harbored bft‐3 isotype (16.66%). These bacteria harbor antimicrobial resistance genes that could be transferred to other susceptible intestinal strains. Further investigations on lineage analysis are needed for a better understanding of these bacteria in Iran.     

Investigation of Acinetobacter baumannii resistance to carbapenems in Marseille hospitals,south of France: a transition from an epidemic to an endemic situation

Carbapenem‐resistant Acinetobacter baumannii infections are a worldwide endemic nosocomial threat. Between December 2010 and April 2011, an increase of carbapenem‐resistant A. baumannii infections occurred in several Marseille University Hospitals. The aim of this study was to investigate the increase of carbapenem‐resistant A. baumannii infections and to characterize the mechanisms of carbapenem resistance. The increase was detected by a homemade computer surveillance program, known as EPIMIC, that monitors antibiotic resistance profiles on a weekly basis. During this period, positive samples of carbapenem‐resistant A. baumannii were retrieved from patients hospitalized in different units. Genotyping of the isolates was performed using pulsed‐field gel electrophoresis (PFGE) and multi‐locus sequence typing (MLST), and carbapenemase gene analyses were performed to detect the presence of carbapenemases and to determine the relationships of the isolates. Carbapenem‐resistant A. baumannii were isolated in a total of 11 patients who were hospitalized in different hospitals units. We identified the presence of the bla_OXA23‐like carbapenemase‐encoding gene in all of the isolates and found four major PFGE groups and different MLST groups. These results demonstrate a current evolution in the A. baumannii epidemiology in Marseille with a switch from an epidemic situation to an endemic situation and with several circulating clones.     

Survival of Campylobacter jejuni and Campylobacter coli water isolates in lake and well water

The role of water for transmission of Campylobacter jejuni and C. coli to humans might be underestimated, as factors important for bacterial viability in water are largely unknown. We have studied water survival of seven C. jejuni and eight C. coli isolates originally isolated from Swedish waters, together with selected reference strains, over eight days at 4 °C in the dark in untreated water collected from a local lake and a private well. To study seasonality, lake water samples were collected during spring and autumn. Samples for culturable bacterial counts were taken on days 2, 4, 6, and 8 and compared to the start inoculum. For C. jejuni, a significantly better survival was observed in autumn than in spring lake water. Furthermore, C. jejuni had a significantly better survival than C. coli in autumn lake and well water samples; the rate of culturability loss was almost double for C. coli in autumn lake water. These findings contribute to a better understanding on the seasonality of waterborne Campylobacter infections and the general predominance of C. jejuni.     

Challenges in the identification of methicillin‐resistant Staphylococcus argenteus by routine diagnostics

Current clinical diagnostic procedures have shortcomings in the differentiation of Staphylococcus argenteus from Staphylococcus aureus. This article presents three cases of Staphylococcus argenteus obtained from clinical samples. The initial results from biochemical and molecular methods led to an incorrect identification of the isolates as methicillin‐resistant Staphylococcus aureus. Whole genome sequencing and real‐time PCR targeting the nonribosomal peptide synthetase gene led to their correct identification as methicillin‐resistant Staphylococcus argenteus. The study shows that real‐time PCR can be used to differentiate the two species in routine diagnostics. For purposes of identification based on whole genome sequencing, the MinION portable sequencer is a simple and affordable approach which could be used by many laboratories.     

Decreased susceptibility to chlorhexidine and prevalence of disinfectant resistance genes among clinical isolates of Staphylococcus epidermidis

Staphylococcus epidermidis is a versatile agent, being both a commensal and a nosocomial pathogen usually with an opportunistic role in association with implanted foreign body materials. Pre‐operative antiseptic preparation is an important strategy for reducing the risk of complications such as surgical site infection (SSI). Currently, the most widely used antiseptics are alcohols, quaternary ammonium compounds (QACs), and the bisbiguanide chlorhexidine. Occurrence of resistance to the latter agent has drawn increasing attention. The aim of this study was to investigate if decreased susceptibility to chlorhexidine among S. epidermidis was present in our setting, a Swedish university hospital. Staphylococcus epidermidis (n = 143), retrospectively collected, were obtained from prosthetic joint infections (PJI) (n = 61), post‐operative infections after cardiac surgery (n = 31), and the skin of the chest after routine disinfection prior to cardiac surgery (n = 27). In addition, 24 commensal isolates were included. Minimum inhibitory concentration (MIC) of chlorhexidine was determined on Mueller Hinton agar plates supplemented with serial dilutions of chlorhexidine. Five QAC resistance genes, qacA/B, smr, qacH, qacJ, and qacG, were detected using PCR. Decreased susceptibility to chlorhexidine was found in 54% of PJI isolates, 68% of cardiac isolates, 21% of commensal isolates, and 7% of skin isolates from cardiac patients, respectively. The qacA/B gene was present in 62/143 isolates (43%), smr in 8/143 (6%), and qacH in one isolate (0.7%). The qacA/B gene was found in 52% of PJI isolates, 61% of cardiac isolates, 25% of commensal isolates, and 19% of the skin isolates. In conclusion, decreased susceptibility to chlorhexidine, as well as QAC resistance genes, were prevalent among S. epidermidis isolates associated with deep SSIs.