Repetitive Extragenic Palindromic Polymerase Chain Research Articles

Unlocking the diversity and plant‐growth promoting potential of the bulk soil bacteria associated with the Saharan tree Vachellia tortilis subsp. raddiana

AbstractIn this study, we investigated the bacterial diversity and plant growth‐promoting (PGP) properties of bulk soil bacteria of Vachellia tortilis subsp. raddiana collected from the Taidalt and Amazloug nature preserves area in southern Morocco. Using repetitive extragenic palindromic polymerase chain reaction fingerprinting and 16S rDNA sequencing, 93 strains were identified. They belonged mainly to the genera Bacillus and Peribacillus. In vitro tests for PGP traits showed high levels of activity for strains belonging to the genera Bacillus and Pseudomonas. It was relevant that several Bacillus strains produced auxin (maximum amount 262.61 µg mL−1 for Bacillus sp. LMR1097), solubilized phosphate (maximum amount 22.10 µg mL−1), or produced siderophores, while three strains were able to fix atmospheric nitrogen (LMR1145, LMR1013, and LMR1015). Inoculation of V. tortilis subsp. raddiana plants with selected bacterial strains (LMR881 and LMR1097) increased shoot and root growth parameters. The strain Pantoea sp. LMR881 had the highest mean values for shoot and root length (24.60–14.80 cm) and shoot dry weight (1.31 g), whereas Bacillus sp. LMR1097 showed the lowest mean shoot dry weight. Overall, these results highlight the potential of using selected native bacterial inoculants for improving growth of candidate tree plants to be used in restoration programs of arid degraded areas.

Outbreak of Burkholderia multivorans among patients at two acute-care hospitals in California, August 2021–July 2022

Background:Burkholderia multivorans are gram-negative bacteria typically found in water and soil. B. multivorans outbreaks among patients without cystic fibrosis have been associated with exposure to contaminated medical devices or nonsterile aqueous products. Acquisition can also occur from exposure to environmental reservoirs like sinks or other hospital water sources. We describe an outbreak of B. multivorans among hospitalized patients without cystic fibrosis at 2 hospitals within the same healthcare system in California (hospitals A and B) between August 2021 and July 2022. Methods: We defined confirmed case patients as patients without cystic fibrosis hospitalized at hospital A or hospital B between January 2020 to July 2022 with B. multivorans isolated from any body site matching the outbreak strain. We reviewed medical records to describe case patients and to identify common exposures. We evaluated infection control practices and interviewed staff to detect exposures to nonsterile water. Select samples from water, ice, drains, and sink splash zone surfaces were collected and cultured for B. multivorans in March 2022 and July 2022 from both hospitals. Common aqueous products used among case patients were tested for B. multivorans. Genetic relatedness between clinical and environmental samples was determined using random amplified polymorphic DNA (RAPD) and repetitive extragenic palindromic polymerase chain reaction (Rep-PCR). Results: We identified 23 confirmed case patients; 20 (87%) of these were identified at an intensive care unit (ICU) in hospital A. B. multivorans was isolated from respiratory sources in 18 cases (78%). We observed medication preparation items, gloves, and patient care items stored within sink splash zones in ICU medication preparation rooms and patient rooms. Nonsterile water and ice were used for bed baths, swallow evaluations, and ice packs. B. multivorans was cultured from ice and water dispensed from an 11-year-old ice machine in the ICU at hospital A in March 2022 but no other water sources. Additional testing in July 2022 yielded B. multivorans from ice and a drain pan from a new ice machine in the same ICU location at hospital A. All products were negative. Clinical and environmental isolates were the same strain by RAPD and Rep-PCR. Conclusions: The use of nonsterile water and ice from a contaminated ice machine contributed to this outbreak. Water-related fixtures can serve as reservoirs for Burkholderia, posing infection risk to hospitalized and immunocompromised patients. During outbreaks of water-related organisms, such as B. multivorans , nonsterile water and ice use should be investigated as potential sources of transmission and other options should be considered, especially for critically ill patients.Disclosures: None

Hospital-acquired infections due to carbapenem-resistant Providencia stuartii.

During the course of a retrospective survey on healthcare associated infections (HAIs) due to carbapenem-resistant organisms, an unusual prevalence of HAIs due to carbapenem-resistant Providencia stuartii (CRPS) was found. Hence this study aimed to conduct the occurrence of P. stuartii associated HAIs with special reference to the drug resistance profiling of these isolates. Of the eight total HAI cases (7.5% of total HAIs and 33.3% of HAIs due to Enterobacterales) of CRPS infections included in this study, three were reported from ventilator-associated pneumonia (VAP), three were surgical site infections (SSIs), one was a catheter-associated urinary tract infection (CAUTI) and one was a bloodstream infection. All the eight CRPS isolates were tested for extended-spectrum β-lactamases production, AmpC hyperproduction as well as carbapenem resistance. Typing of the isolates was performed by repetitive extragenic palindromic polymerase chain reaction (REP-PCR). All the eight isolates of CRPS were found to be AmpC hyperproducers, carbapenemase producers, and harboured chromosomally located blaNDM in seven isolates and blaIMP genes in one. All the cases with CRPS infections had prior history of colistin therapy along with prolonged hospital stay (>20 days). The cases were located in five different wards/intensive care unit (ICU) within the hospital in one year. However, strain typing by REP-PCR revealed 100 per cent similarity and clonal relatedness in all the seven isolates carrying blaNDM genes. Interestingly, routine hospital surveillance revealed a high carriage of P. stuartii in the axilla of patients admitted to the ICU. The study findings suggest CRPS as an important cause of HAIs. This organism often goes unnoticed due to the burden of carbapenem resistance in other Enterobacterales and non-fermenters.

Genetic relationship, antibiotic resistance pattern and virulence factors of Klebsiella pneunoniae strains isolated from meningitis patients

Backgroud: Meningitis due to Klebsiella pneunoniae is increasingly reported from several Asian countries. Microbiological characteristic of K. pneumoniae strains causing meningitis is not yet explored in Iran. The aim of this study was to demonstrate antibiotic resistance pattern and virulence factors, as well as the genetic relationship of K. pneumoniae strains isolated from meningitis patients. Materials and Mehtods: Eight K. pneumoniae isolates were collected from hospitalized patients at Imam Khomeini hospital, Tehran-Iran from 14 May 2018 to 15 Mar 2020. The antibiotic resistance pattern was determined by disc diffusion method. Antibiotic resistance genes and virulence-associated genes were traced by Polymerase Chain Reaction. Repetitive Extragenic Palindromic Polymerase Chain Reaction was used for evaluation of the genetic relationships among isolates. Findings: Six out of eight isolates were resistant to almost all the 15 antibiotics tested. These six isolates harbored triple antibiotic resistance genes: blaOXA-48, blaSHV, and aac (6’)-Ib. Five of these isolates co-harbored aac (3)-IIa gene. Besides, blaTEM and blaCTXM-1 were detected in five and three isolates respectively. blaNDM-1 was identified in one isolate. All isolates harbored acrAB, ompK36, and tolC resistance genes. None of the isolates were related to K1 or K2 capsular serotypes. The most commonly detected virulence genes were entB (100%), mrkD (100%), ybtS (62.5%) and kfu (25%). rep-PCR fingerprinting discriminated seven isolates into three clusters. Conclusion: Simulations presence of the genes coding for Extended Spectrum Beta Lactamases, Carbapenemases, and Aminoglycoside Modifying Enzymes narrows therapeutic alternatives and imposes a heavy load on the public health system. Antibiotic susceptibility test is recommended however, Carbapenems can still be considered as first-line medications for preliminary empirical treatment of K. pneumoniae meningitis before obtaining susceptibility test results. Low genetic diversity suggests the circulation of certain K. pneumoniae clones in hospital which highlights the establishing of effective infection observation and prevention program.

Mycobacterium salmoniphilum and M. chelonae in Captive Populations of Chinook Salmon.

Chinook Salmon Oncorhynchus tshawytscha is a keystone fish species in the Pacific Northwest. In 2019, unusual mortalities occurred in two different populations of cultured fingerlings from the same facility in California, USA. The systems consist of outdoor, enclosed, flow-through freshwater tanks that are maintained at 18±1°C. Clinical signs and gross findings were only observed in one population and included abnormal swimming, inappetence, lethargy, skin discoloration, and the presence of multifocal nodular and ulcerative skin lesions. Microscopic lesions were infrequent and consisted of severe, locally extensive granulomatous dermatitis and myositis and mild, multifocal, granulomatous branchitis, myocarditis, and hepatitis. Intracellular acid-fast organisms were observed within areas of granulomatous myositis. Posterior kidney swabs were collected and inoculated in nutrient-rich and selective agar media and incubated at 25°C for 2weeks. Visibly pure bacterial colonies were observed 7-10 d postinoculation. Partial sequences of 16S rRNA initially identified the recovered bacteria as members of the genus Mycobacterium. However, marked variability was observed among Mycobacterium spp. isolates by using repetitive extragenic palindromic polymerase chain reaction fingerprinting. Amplification and sequencing of the ribosomal RNA internal transcribed spacer, 65-kDa heat shock protein, and RNA polymerase β-subunit gene of the cultured isolates identified M. salmoniphilum and M. chelonae, discrete members of the M. chelonae-abscessus complex, isolated from diseased Chinook Salmon fingerlings.

Assessment of genetic relatedness among commercial and wild strains of Agaricus bisporus using repetitive extragenic palindromic sequences and polymerase chain reaction

Abstract The genetic relatedness among 18 strains of Agaricus bisporus was assessed based on the fragment pattern analysis obtained by the amplification of genomic DNA by BOX, ERIC (ERICIR-I/ERIC2) and REP (REP1RI/REP2I) gene sequences. Based on the banding patterns of PCR-amplified products, eight putative groups among the 18 commercial and wild strains were recognized. REP-PCR generated multiple distinct products showing considerable variability among the strains with ERIC and REP elements successfully enabled detection of wild and commercial A. bisporus. Strains originating from the same geographical location were not always genetically related. To our knowledge, this was the first relevance study of biodiversity in commercial and native populations of A. bisporus by using the REP-PCR technique. The results confirmed the usefulness REP-PCR typing in intraspecific genetic variation assessments of the button mushroom. High level of Iranian wild strains distance with the commercial cultivars approves their importance as a promising new source of diversity in A. bisporus breeding program.

Presence of Extended-Spectrum β-lactamase, CTX-M-65 in Salmonella enterica serovar Infantis Isolated from Children with Diarrhea in Lima, Peru

AbstractGastroenteritis in children is a serious condition in many parts of the world. Salmonella enterica is one of the causes of the disease. In this study, 280 fecal samples from children with diarrhea in four hospitals in Lima, Peru, were collected between September 2012 and March 2013. Salmonella was detected in 26 of the samples. Serotyping demonstrated that 25 of the isolates were S. enterica Infantis, and one isolate was S. enterica Typhimurium. Repetitive extragenic palindromic–polymerase chain reaction analysis suggests that all S. Infantis belong to the same clone. All but one of the S. Infantis isolates exhibited an extended-spectrum β-lactamase phenotype as they harbored bla CTX-M 65. Two strains also carried bla TEM-1. Nine of the isolates were resistant to azithromycin and two to ciprofloxacin. This study demonstrates that a multidrug-resistant S. Infantis clone carrying bla CTX-M 65 was circulating among children in Lima, Peru. The development of molecular epidemiology studies in Salmonella-causing diarrhea or other pathologies in Lima and in other areas will be useful to determine the permanence, geographical spread, and clinical implications of this clone.

Continuous Flow-Constructed Wetlands for the Treatment of Swine Waste Water.

The microbiological quality of treated waste water is always a concern when waste water is disposed to the environment. However, when treated appropriately, such water can serve many purposes to the general population. Therefore, the treatment and removal of contaminants from swine waste water by continuous flow-constructed wetlands involves complex biological, physical, and chemical processes that may produce better quality water with reduced levels of contaminants. Swine waste contains E. coli populations and other bacterial contaminants originating from swine houses through constructed wetlands, but little is known about E. coli population in swine waste water. To assess the impacts of seasonal variations and the effect of the wetland layout/operations on water quality, E. coli isolates were compared for genetic diversity using repetitive extragenic palindromic polymerase chain reaction (REP-PCR). None of the isolates was confirmed as Shiga toxin producing E. coli O157:H7 (STEC); however, other pathotypes, such as enterotoxigenic E. coli (ETEC) were identified. Using a 90% similarity index from REP-PCR, 69 genotypes out of 421 E. coli isolates were found. Our data showed that the E. coli population was significantly (p = 0.036) higher in November than in March and August in most of the wetland cells. Furthermore, there was a significant (p = 0.001) reduction in E. coli populations from wetland influent to the final effluent. Therefore, the use of continuous flow-constructed wetlands may be a good treatment approach for reducing contaminants from different waste water sources.

Molecular Analysis of Shigella sonnei Strains Isolated from Hospitals of Tehran and Ilam Cities Using Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR)

Molecular Analysis of Shigella sonnei Strains Isolated from Hospitals of Tehran and Ilam Cities Using Repetitive Extragenic Palindromic Polymerase Chain Reaction (REP-PCR)

Molecular Typing of Klebsiella pneumoniae isolates using Repetitive Extragenic Palindromic Sequence-Based PCR in a Hospital in Tehran, Iran

Background: The presence of extended-spectrum β-lactamases (ESBLs) is increasing worldwide and blaCTX-M1 is the predominant β-lactamase. Objective: This study was conducted to determine the ESBL production and prevalence of blaCTX-M1, blaSHV and blaTEM and AmpC genes and repetitive extragenic palindromic polymerase chain reaction (rep-PCR) pattern among Klebsiella pneumoniae isolates in Tehran from 2014 to 2016. Materials and Methods: One hundred eleven isolates were collected during the study period. The PCR was employed to detect the blaCTX-M1, blaSHV, blaTEM and AmpC genes. The genetic relation of isolates was performed using rep-PCR typing method. Results: Eighty-three and 86 isolates showed Minimum inhibitory concentration (MIC) ≥2 against ceftazidime and cefotaxime, respectively and 80 (72%) isolates exhibited ESBL production. The prevalence of blaCTX-M1, blaSHV, blaTEM and AmpC genes among ESBL producers was 92.5% (n = 74), 66.2% (n = 53), 56.2% (n = 45) and 2.5% (n = 2), respectively. The rep-PCR typing pattern of isolates showed a wide diversity, indicating the polyclonal spread of CTX-M type producing isolates. Conclusion: The findings of this study highlighted the emergence and spread of K. pneumoniae isolates producing CTX-M and other ESBL enzymes with diverse genetic backgrounds in a hospital in Tehran.

Mingling of human and veterinary strains of Staphylococcus aureus: An emerging issue in health-care systems

Aim: Methicillin-resistant Staphylococcus aureus remains a leading cause of hospital and community infections. We report a retrospective molecular characterization of S. aureus strains from different settings: hospital workers and patients, and veterinarian surgeons and pets. Materials and Methods: Eighty-nine S. aureus isolates obtained from nasal swabs of 10 patients, 17 health-care workers (HCWs), 9 pets, and 53 veterinarians were genotypically characterized by means of repetitive extragenic palindromic polymerase chain reaction (Rep PCR) and whole-genome sequencing. Results: Thirteen different sequence types (STs) were detected: ST398, ST22, ST8, ST30, ST15, ST5, ST121, ST45, ST10, ST6, ST34, ST97, and ST1. Two new STs differing from ST22 and ST5 for a single multilocus sequence typing gene were also identified. Rep PCR documented a genetic relationship among isolates obtained from 5 veterinarians and 10 HCWs. Conclusion: The large diversity of S. aureus strains detected may reflect a larger epidemiology within the hospital and community, in which companion animals likely act as a reservoir. We identified the circulation of ST5, ST8, ST15, ST22, ST30, ST45, and ST121 both in the hospital and veterinarian environment. Starting from the idea of a unique setting where our population lives, we consider the relationship between community- and hospital-acquired S. aureus.

In vitro activity of colistin mono- and combination therapy against colistin-resistant Acinetobacter baumannii, mechanism of resistance, and clinical outcomes of patients infected with colistin-resistant A. baumannii at a Thai university hospital.

PurposeColistin is a drug of last resort for treating multidrug-resistant Acinetobacter baumannii infections. Unfortunately, colistin-resistant A. baumannii (CoR-AB) has been reported. Here, we examined the in vitro effect of mono- and combined antimicrobials against CoR-AB strains and their resistance mechanism, and evaluated the clinical outcomes of CoR-AB-infected patients.Patients and methodsSeventeen clinical CoR-AB strains were isolated from patients at Phramongkutklao hospital, 2011–2015. The mono- and synergistic activities of colistin, tigecycline, sulbactam, imipenem, meropenem, amikacin, fosfomycin, and cotrimoxazole were examined by minimum inhibitory concentration (MIC) and fractional inhibitory concentration index. Clonal relationship and resistance genes were determined by repetitive extragenic palindromic polymerase chain reaction with specific primers. The effect of carbonyl cyanide 3-chlorophenylhydrazone combined with colistin was used to test efflux pump involvement. Patient treatment outcomes were also reported.ResultsThe most prevalent infection in CoR-AB patients was pneumonia (35.3%), and all patients were administered colistin combined with another agent. The 30-day mortality was 70.6%, and the colistin MIC range and MIC50 was 16–512 μg/mL and 64 μg/mL, respectively. All CoR-AB strains were sensitive to tigecycline. Sporadic isolates were susceptible to sulbactam, imipenem, meropenem, and cotrimoxazole. A synergistic or additive effect was observed for colistin plus imipenem or meropenem (16.7%), sulbactam (66.7%), or tigecycline (66.7%). The CoR-AB isolates could be divided into four different clones (A–D) with a high prevalence of group B (47.1%). Eight isolates harbored blaOXA23, blaIMP, blaKPC, and blaNDM, and one contained blaOXA23, blaIMP, and blaKPC, while the eight remaining isolates carried only blaOXA23. The MIC values of all strains were greatly reduced for colistin plus carbonyl cyanide 3-chlorophenylhydrazone.ConclusionCoR-AB clinical isolates exhibited very high colistin resistance and a high frequency of resistance genes. The mechanism of colistin resistance appears to be mediated via an efflux pump. Thus, certain antimicrobials could be used as salvage therapy for CoR-AB infection.

Whole genome sequencing for the molecular characterization of carbapenem-resistant Klebsiella pneumoniae strains isolated at the Italian ASST Fatebenefratelli Sacco Hospital, 2012\u20132014

BackgroundThe emergence of carbapenem-resistant Klebsiella pneumoniae strains is threatening antimicrobial treatment.MethodsSixty-eight carbapenemase-producing K. pneumoniae strains isolated at Luigi Sacco University Hospital-ASST Fatebenefratelli Sacco (Milan, Italy) between 2012 and 2014 were characterised microbiologically and molecularly. They were tested for drug susceptibility and carbapenemase phenotypes, investigated by means of repetitive extra-genic palindromic polymerase chain reaction (REP-PCR), and fully sequenced by means of next-generation sequencing for the in silico analysis of multi-locus sequence typing (MLST), their resistome, virulome and plasmid content, and their core single nucleotide polymorphism (SNP) genotypes.ResultsAll of the samples were resistant to carbapenems, other β-lactams and ciprofloxacin; many were resistant to aminoglycosides and tigecycline; and seven were resistant to colistin. Resistome analysis revealed the presence of blaKPC genes and, less frequently blaSHV, blaTEM, blaCTX-M and blaOXA, which are related to resistance to carbapenem and other β-lactams. Other genes conferring resistance to aminoglycoside, fluoroquinolone, phenicol, sulphonamide, tetracycline, trimethoprim and macrolide-lincosamide-streptogramin were also detected. Genes related to AcrAB-TolC efflux pump-dependent and pump-independent tigecycline resistance mechanisms were investigated, but it was not possible to clearly correlate the genomic features with tigecycline resistance because of the presence of a common mutation in susceptible, intermediate and resistant strains. Concerning colistin resistance, the mgrB gene was disrupted by an IS5-like element, and the mobile mcr-1 and mcr-2 genes were not detected in two cases. The virulome profile revealed type-3 fimbriae and iron uptake system genes, which are important during the colonisation stage in the mammalian host environment. The in silico detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX1, IncX3, IncFII(K), IncN, IncL/M(pMU407) and IncFIA(HI1). REP-PCR showed five major clusters, and MLST revealed six different sequence types: 512, 258, 307, 1519, 745 and 101. Core SNP genotyping, which led to four clusters, correlated with the MLST data. Isolates of the same sequencing type often had common genetic traits, but the SNP analysis allowed greater strain tracking and discrimination than either the REP-PCR or MLST analysis.ConclusionOur findings support the importance of implementing bacterial genomics in clinical medicine in order to complement traditional methods and overcome their limited resolution.

A Prospective Observational Study of the Epidemiology, Management, and Outcomes of Skin and Soft Tissue Infections Due to Carbapenem-Resistant Enterobacteriaceae.

BackgroundThis study was performed to characterize the epidemiology, management, and outcomes of skin and soft tissue infection (SSTI) and colonization due to carbapenem-resistant Enterobacteriaceae (CRE).MethodsPatients from the Consortium on Resistance Against Carbapenem in Klebsiella and Other Enterobacteriaceae (CRACKLE-1) from December 24, 2011 to October 1, 2014 with wound cultures positive for CRE were included in the study. Predictors of surgical intervention were analyzed. Molecular typing of isolates was performed using repetitive extragenic palindromic polymerase chain reaction (PCR). Carbapenemase genes were detected using PCR.ResultsOne hundred forty-two patients were included: 62 had SSTI (44%) and 56% were colonized. Mean age was 61 years, and 48% were male: median Charlson score was 3 (interquartile range, 1–5). Forty-eight percent of patients were admitted from long-term care facilities (LTCFs), and 31% were from the community. Two strain types (ST258A and ST258B) were identified (73% of 45 tested). Carbapenemase genes were detected in 40 of 45 isolates (blaKPC-3 [47%], blaKPC-2 [42%]). Sixty-eight patients (48%) underwent surgical intervention, 63% of whom had SSTI. Patients admitted from LTCFs were less likely to undergo surgical intervention (odds ratio [OR], 0.36; 95% confidence interval [CI], 0.18–0.71). In multivariable analysis, among patients with SSTI, those admitted from LTCFs were less likely to undergo debridement (OR, 0.18; 95% CI, 0.04–0.93).ConclusionsPatients admitted from LTCFs with CRE SSTI were less likely to undergo surgical intervention. Sixteen percent of the patients died, and approximately 50% of survivors required more intensive care upon discharge. These findings suggest a unique, impactful syndrome within the CRE infection spectrum. Further studies are needed to assess the role of surgical debridement in management of CRE-SSTI, particularly among LTCF residents.

An Outbreak of Burkholderia cepacia Complex Infections Associated with Contaminated Liquid Docusate.

OBJECTIVE To investigate an outbreak of Burkholderia cepacia complex and describe the measures that revealed the source. SETTING A 629-bed, tertiary-care, pediatric hospital in Houston, Texas. PATIENTS Pediatric patients without cystic fibrosis (CF) hospitalized in the pediatric and cardiovascular intensive care units. METHODS We investigated an outbreak of B. cepacia complex from February through July 2016. Isolates were evaluated for molecular relatedness with repetitive extragenic palindromic polymerase chain reaction (rep-PCR); specific species identification and genotyping were performed at an independent laboratory. The investigation included a detailed review of all cases, direct observation of clinical practices, and respiratory surveillance cultures. Environmental and product cultures were performed at an accredited reference environmental microbiology laboratory. RESULTS Overall, 18 respiratory tract cultures, 5 blood cultures, 4 urine cultures, and 3 stool cultures were positive in 24 patients. Among the 24 patients, 17 had symptomatic infections and 7 were colonized. The median age of the patients was 22.5 months (range, 2-148 months). Rep-PCR typing showed that 21 of 24 cases represented the same strain, which was identified as a novel species within the B. cepacia complex. Product cultures of liquid docusate were positive with an identical strain of B. cepacia complex. Local and state health departments, as well as the CDC and FDA, were notified, prompting a multistate investigation. CONCLUSIONS Our investigation revealed an outbreak of a unique strain of B. cepacia complex isolated in clinical specimens from non-CF pediatric patients and from liquid docusate. This resulted in a national alert and voluntary recall by the manufacturer. Infect Control Hosp Epidemiol 2017;38:567-573.

The influence of biofilm formation and multidrug resistance on environmental survival of clinical and environmental isolates of Acinetobacter baumannii

Acinetobacter baumannii is a gram-negative, opportunistic pathogen. Its ability to form biofilm and increasing resistance to antibiotic agents present challenges for infection control. A better understanding of the influence of biofilm formation and antibiotic resistance on environmental persistence of A baumannii in hospital settings is needed for more effective infection control. A baumannii strains isolated from patients and the hospital environment were identified via Matrix Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Bellerica, MA), repetitive extragenic palindromic polymerase chain reaction genotyped, and antibiotic resistance was determined using Vitek 2 (bioMérieux, Inc, Durham NC). Biofilm mass was quantified via microtiter plate method and desiccation tolerance determined up to 56 days. High biofilm forming, clinical, multidrug-resistant- (MDR) positive strains were 50% less likely to die of desiccation than low biofilm, non-MDR strains. In contrast, environmental, MDR-positive, low biofilm forming strains had a 2.7 times increase in risk of cell death due to desiccation compared with their MDR-negative counterparts. MDR-negative, high biofilm forming environmental strains had a 60% decrease in risk compared with their low biofilm forming counterparts. The MDR-positive phenotype was deleterious for environmental strains and the high biofilm phenotype was critical for survival. This study provides evidence of the trade-off between antibiotic resistance and desiccation tolerance, driven by condition-dependent adaptation, and establishes rationale for research into the genetic basis of the variation in fitness cost between clinical and environmental isolates.

The Complex Epidemiology of Carbapenem-Resistant Enterobacter Infections: A Multicenter Descriptive Analysis.

The pandemic of carbapenem-resistant Enterobacteriaceae (CRE) was primarily due to clonal spread of bla KPC producing Klebsiella pneumoniae. Thus, thoroughly studied CRE cohorts have consisted mostly of K. pneumoniae. To conduct an extensive epidemiologic analysis of carbapenem-resistant Enterobacter spp. (CREn) from 2 endemic and geographically distinct centers. CREn were investigated at an Israeli center (Assaf Harofeh Medical Center, January 2007 to July 2012) and at a US center (Detroit Medical Center, September 2008 to September 2009). bla KPC genes were queried by polymerase chain reaction. Repetitive extragenic palindromic polymerase chain reaction and pulsed-field gel electrophoresis were used to determine genetic relatedness. In this analysis, 68 unique patients with CREn were enrolled. Sixteen isolates (24%) were from wounds, and 33 (48%) represented colonization only. All isolates exhibited a positive Modified Hodge Test, but only 93% (27 of 29) contained bla KPC. Forty-three isolates (63%) were from elderly adults, and 5 (7.4%) were from neonates. Twenty-seven patients died in hospital (40.3% of infected patients). Enterobacter strains consisted of 4 separate clones from Assaf Harofeh Medical Center and of 4 distinct clones from Detroit Medical Center. In this study conducted at 2 distinct CRE endemic regions, there were unique epidemiologic features to CREn: (i) polyclonality, (ii) neonates accounting for more than 7% of cohort, and (iii) high rate of colonization (almost one-half of all cases represented colonization). Since false-positive Modified Hodge Tests in Enterobacter spp. are common, close monitoring of carbapenem resistance mechanisms (particularly carbapenemase production) among Enterobacter spp. is important.

Assessment of two multilocus sequence typing (MLST) schemes available for Streptococcus mutans

ObjectiveTwo multilocus sequencing typing (MLST) schemes are currently available for Streptococcus mutans. The first, introduced by Nakano et al. in 2007, consists of 8 conserved housekeeping genes. The second, introduced in 2010 by Do et al., includes 6 housekeeping genes and 2 putative virulence genes. The purpose of the current study was to compare the two MLST schemes for use in validating repetitive extragenic palindromic polymerase chain reaction (rep-PCR) genotypes. DesignThirty-three S. mutans isolates, representing the 11 most commonly occurring rep-PCR genotype groups, were selected for MLST. MLST was performed with SYBR Green™ PCR with published primers for both MLST schemes. Amplicons were purified, sequenced, and data checked against the www.PubMLST.org database for allelic and sequence type (ST) assignment. Discriminatory power, congruence, and convenience criteria were evaluated. Concatenated sequences for each scheme were analyzed using MEGA to generate phylogenetic trees using minimum evolution with bootstrap. ResultsNo significant difference in discriminatory power was observed between the two MLST schemes for S. mutans. Clonal clusters were consistent for both schemes. Overall, MLST demonstrated marginally greater discriminatory power than rep-PCR; however all methods were found to be congruent. New alleles and ST are reported for each scheme and added to the PubMLST database. ConclusionsClonality, supported by both methods and rep-PCR, indicates S. mutans genotypes are shared between unrelated subjects. Both Nakano and Do schemes demonstrates similar genotype discrimination for S. mutans isolates suggesting each are well designed and may be used to verify rep-PCR genotypes.

A gloves-associated outbreak of imipenem-resistant Acinetobacter baumannii in an intensive care unit in Guangdong, China.

BackgroundImipenem-resistant Acinetobacter baumannii (IRAB) is an important cause of hospital-acquired infection. We aimed to describe an outbreak of IRAB infection and to investigate its possible source in an intensive care unit.MethodsAn environmental investigation was undertaken. Antimicrobial susceptibility testing was performed by microdilution. These isolates were genotyped by use of repetitive extragenic palindromic polymerase chain reaction (rep-PCR; DiversiLab). The study included 11 patients infected with IRAB and 14 control patients free of IRAB. Case and control patients were compared for possible predisposing factors. A multifaceted intervention was implemented to control the outbreak.ResultsThirty-nine IRABs were isolated from patients and the environmental surveillance culture in August, November, and December 2011. All isolates were resistant to imipenem. The IRAB strains belonged to seven clones (A–G) by the use of rep-PCR. There were four epidemic clones (D–G) in the outbreak, and Clone D was predominant. For the case–control study, patients with chronic obstructive pulmonary disease were susceptible to infection with IRAB. The hospital mortality of the case group was significantly higher than that of the control group.ConclusionsThe outbreak strains were transmitted among infected patients and equipment by inappropriate use of gloves. A combination of aggressive infection control measures is essential for preventing recurrent nosocomial outbreaks of IRAB.

Utility of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for the rapid detection of nosocomial outbreaks of multidrug resistant organisms: Experience at a tertiary care center in North India

There is a huge need to develop molecular typing methods which are simple to perform, rapid and cost effective to confirm clonality of nosocomial isolates in outbreak situations. The aim of the study was to investigate a hospital outbreak of multi-drug resistant (MDR) Klebsiellapneumoniae septicemia in a paediatric surgery intensive care unit (PSICU) using a repetitive extragenic palindromic polymerase chain reaction (REP-PCR). MDR Klebsiella pneumoniae isolates from an outbreak of nosocomial sepsis were typed byREP-PCR using consensus primers. Isolates from different intensive care units (ICUs) but with similar antibiogram were also genotyped for comparison. A cluster of twelve MDR K Pneumoniae septicemia cases was identified at the PSICU by genotyping using REP-PCR. Surveillance cultures failed to pick up any source of infection. REP-PCR was found to be a rapid and simple tool for investigation outbreaks in hospitals. Due to early detection we could initiate infection control practices with focus on hand washing and prevent the further transmission of the organism.