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Abstract This study investigated 247 Escherichia coli isolates collected from four cattle farms to characterize aminoglycoside-modifying enzyme (AME) genes, their plasmid replicons and transferability. Out of 247 isolates a high number of isolates (total 202; 81.78%) were found to be resistant to various antibiotics by disc diffusion. Of the 247 strains, 139 (56.3%) were resistant to streptomycin, and other antibiotic resistances followed as tetracycline (12.15%), ampicillin (7%), chloramphenicol (5.7%) and trimethoprim-sulfamethoxazole (0.8%). Among 247 isolates B1 was the predominant phylogenetic group identified comprising 151 isolates (61.1%), followed by groups A (27.9%), D (7%) and B2 (4%). Out of 139 isolates investigated for AME, 130 (93.5%) isolates carried at least one AME gene. aph3″-1a and aph3″-1b (46%) were the principal genes detected, followed by aac3-IVa (34.5%). ant2″-1a was the least detected gene (2.2%). Nine (6.5%) strains carried no AME genes. Twelve (63.2%) among 19 isolates transferred an AME gene to a recipient and aph3΄-1a was the dominant transferred gene. Transferability mainly occurred via the IncFIB replicon type (52.6%). Pulsed-field gel electrophoresis typing demonstrated a higher degree of diversity with 14 distinct cluster types. This result suggests that commensal microflora from food-producing animals has a tremendous ability to harbor and transfer AME genes, and poses a potential risk by dissemination of resistance to humans through the food chain. This work demonstrated diverse aminoglycoside-modifying enzyme (AME) genes with an ability to transfer via a conjugative plasmid and the role of commensal E. coli strains in the dissemination of antibiotic resistance and causing a concern in human health and food safety.

Clostridium difficile is an important cause of hospital-associated diarrhea. In this report from the CDC, the U.S. burden of C. difficile infection is estimated at nearly 500,000 cases and 30,000 deaths in 2011, with an increasing burden among nonhospitalized persons. Changes in the epidemiology of Clostridium difficile infections have occurred since the emergence of the North American pulsed-field gel electrophoresis type 1 (NAP1) strain, which has been responsible for geographically dispersed hospital-associated outbreaks. 1 – 3 In the United States, hospitalizations for C. difficile infection among nonpregnant adults doubled from 2000 through 2010 and were projected to continue to increase in 2011 and 2012, especially as laboratories transition to more sensitive C. difficile assays, such as the nucleic acid amplification test (NAAT). 4 – 6 On the basis of data from U.S. death certificates, C. difficile infection is the leading cause of gastroenteritis-associated death . . .

Listeria monocytogenes isolates (n = 81) recovered from ready-to-eat meat-based food products (RTEMP) collected in industrial processing plants and retail establishments were genetically characterized for comparison with those from human clinical cases of listeriosis (n = 49). The aim was to assess RTEMP as a possible food source for human infection. L. monocytogenes was detected in 12.5% of the RTEMP samples, and in some cases, counts were above the European food safety criteria. All isolates were assessed by multiplex PCR for serogroup determination and detection of virulence-associated genes inlA, inlB, inlC, inlJ, plcA, hlyA, actA, and iap. Serogroups IIb and IVb dominated in RTEMP and human isolates, and all were positive for the assessed virulence genes. Antibiotic susceptibility testing by the disk diffusion method revealed a low level of resistance among the isolates. Pulsed-field gel electrophoresis (PFGE) of L. monocytogenes isolates, using restriction enzymes ApaI and AscI, revealed genetic variability and differentiated the isolates in five clusters. Although some pulsed-field gel electrophoresis profiles of particular RTEMP and human isolates seemed to be highly related, exhibiting more than 90% similarity, which suggests a possible common source, in most cases the strains were not genetically or temporally matched. The close genetic relatedness of RTEMP and human listeriosis strains stressed the importance of preventive measure implementation throughout the food chain.

The size distribution of viral DNA in natural samples was investigated in a number of marine, brackish and freshwater environments by means of pulsed field gel electrophoresis (PFGE). The method was modified to work with both water and sediment samples, with an estimated detection limit for individual virus genome size groups of 1-2 x 10⁴ virus-like particles (VLP) mL⁻¹ water and 2-4 x 10⁵ VLP cm⁻³ sediment in the original samples. Variations in the composition and distribution of dominant virus genome sizes were analyzed within and between different habitats that covered a range in viral density from 0.4 x 10⁷ VLP mL⁻¹ (sea water) to 300 x 10⁷ VLP cm⁻³ (lake sediment). The PFGE community fingerprints showed a number of cross-system similarities in the genome size distribution with a general dominance of genomes in the 30-48, 50-70 and 145-200 kb size fractions, and with many of the specific genome sizes detected in all the investigated habitats. However, large differences in community fingerprints were also observed between the investigated sites, and some virus genome sizes were found only in specific biotopes (e.g. lake water), in specific ecosystems (e.g. a particular lake) or even in specific microhabitats (e.g. a particular sediment stratum).

Despite global efforts and widespread vaccination to control whooping cough (pertussis) caused by B. pertussis, the re-emergence of pertussis still is being reported all over the world. Antigenic divergence in B. pertussis virulence factors is one of the reasons of pertussis resurgence, resulting in dissimilarity of local and vaccine strains. In this study, clonal spread and variation of B. pertussis virulence factor in isolated strains from Iranian patients have been analyzed. A total of 100 B. pertussis isolates were obtained from Pertussis Reference Laboratory of Pasteur Institute of Iran. Real-time PCR were performed to confirm the B. pertussis strains. The genomic patterns of B. pertussis strains were analyzed by pulsed-field gel electrophoresis (PFGE). Predominant alleles of local strains were ptxP3, ptxA1, prn2, fim 2-1, fim3-2, and cya2. PFGE results showed 25 patterns clustered into 18 PFGE groups. A few similarities between the circulating isolates, vaccine, and standard strains were obtained. Significantly, 48% of the isolates showed dominant pattern with different allelic profiles from vaccine strains. According to the genomic profiles, the clonal spread was observed among the circulating strains. Predominant virulence factor profile was also comparable with other countries. It may be suggested that strain variation between vaccine and local strains may have an effect on pertussis resurgence in Iran like other parts of the world.

The aim of the present study was to verify whether penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) occurred in Brazil prior to the beginning of the 21st century, and to verify whether ampicillin susceptibility can predict susceptibility to other β-lactams in E. faecalis with this inconsistent phenotype. The presence of polymorphisms in the pbp4 gene and genetic diversity among the isolates were investigated. Of 21 PRASEF analyzed, 5 (23.8%) and 4 (19.0%) were imipenem and piperacillin resistant simultaneously by disk diffusion and broth dilution respectively, contradicting the current internationally accepted standards of susceptibility testing. Sequencing of pbp4 gene revealed an amino acid substitution (Asp-573→Glu) in all PRASEF isolates but not in the penicillin-susceptible, ampicillin-susceptible E. faecalis. Most PRASEF (90.5%) had related pulsed-field gel electrophoresis profiles, but were different from other PRASEF described to date. Results demonstrate that penicillin-resistant, ampicillin-susceptible phenotype was already a reality in the 1990s in E. faecalis isolates in different Brazilian states, and some of these isolates were also imipenem- and piperacillin-resistant; therefore, internationally accepted susceptibility criteria cannot be applied to these isolates. According to pbp4 gene sequencing, this study suggests that a specific amino acid substitution in pbp4 gene found in all PRASEF analyzed is associated with penicillin resistance. Penicillin-resistant, ampicillin-susceptible profile was already a reality in the 1990s in Enterococcus faecalis isolates in different Brazilian states, and polymorphisms in pbp4 gene seems be associated with penicillin resistance in these isolates.

NRC publication: Yes

Between June and September 2010, widespread Italian consumer reports of unusual blue spoilage on fresh dairy products were publicized, resulting in the so-called blue mozzarella event. An inordinately high number of samples from mozzarella and whey cheese products of Italian and German production subsequently tested positive for Pseudomonas fluorescens. The aim of this study was to verify whether a selected P. fluorescens strain was responsible for this apparently unusual event. Molecular characterization of 181 isolated P. fluorescens strains was conducted using a newly optimized pulsed-field gel electrophoresis protocol. Although a high number of pulsotypes was found (132), only four pulsotypes were associated with more than one production plant, and only one German isolate had the same pulsotype as was detected in two Italian plants. This is the only evidence of possible cross-contamination among cheeses from the two countries. The overall results did not support the spread of contamination from German to Italian plants or the presence of one environmental strain that spread in both countries.

A multiplex PCR using targets within the serotype-specific region of the capsular polysaccharide synthesis gene cluster of serotypes K1, K2 and K5 was evaluated using the 77 reference serotype strains of Klebsiella, and a panel of clinical isolates subjected previously to conventional serotyping. The PCR was highly specific for these serotypes, which are those most associated with virulence in humans and horses. PCR confirmed that isolates of the K5 serotype had cross-reacted with antiserum for other serotypes, particularly for K7. K5 isolates received by our laboratory were almost exclusively from thoroughbred horses, and were submitted for screening prior to breeding programmes. Most, including a reference strain isolated in 1955, belonged to a cluster of genetically similar isolates of sequence type (ST) 60. K1 isolates, all from humans, belonged to a previously identified cluster of ST 23.

Background: A carbapenem-resistant Enterobacteriaceae (CRE) outbreak occurred in an advanced emergency medical service center [hereafter referred to as the intensive care unit (ICU)] between 2016 and 2017. Aim: Our objective was to evaluate the infection control measures for CRE outbreaks. Methods: CRE strains were detected in 16 inpatients located at multiple sites. Environmental cultures were performed and CRE strains were detected in 3 of 38 sites tested. Pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and detection of β-lactamase genes were performed against 25 CRE strains. Findings: Molecular typing showed the PFGE patterns of two of four Klebsiella pneumoniae strains were closely related and the same MLST (ST2388), and four of five Enterobacter cloacae strains were closely related and same MLST (ST252). Twenty-three of 25 CRE strains harbored the IMP-1 β-lactamase gene and 15 of 23 CRE strains possessed IncFIIA replicon regions. Despite interventions by the infection control team, new inpatients with the CRE strain continued to appear. Therefore, the ICU was partially closed and the inpatients with CRE were isolated, and the ICU staff was divided into two groups between inpatients with CRE and non-CRE strains to avoid cross-contamination. Although the occurrence of new cases dissipated quickly after the partial closure, a few months were required to eradicate the CRE outbreak. Conclusion: Our data suggest that the various and combined measures that were used for infection control were essential in stopping this CRE outbreak. In particular, partial closure to isolate the ICU and division of the ICU staff were effective.